Acute ischemic cardiac dysfunction is attenuated via gene transfer of a peptide inhibitor of the beta-adrenergic receptor kinase (betaARK1)

Acute myocardial ischemia is a critical adverse effect potentially occurring during cardiac procedures. A peptide inhibitor of the beta-adrenergic receptor kinase (betaARK1), betaARKct, has been successful in rescuing chronic myocardial ischemia. The present study focused on the effects of adenoviral-mediated betaARKct (Adv-betaARKct) delivery on left ventricle (LV) dysfunction induced by acute coronary occlusion. Rabbits received intracoronary delivery of phosphate-buffered saline (PBS) (n=9) or 5x10(11) viral particles of betaARKct (n=8). A loose prolene 5-0 Potz-loop suture was placed around the circumflex coronary artery (LCx) with both ends buried under the skin. Four days later, the suture was retrieved and pulled to occlude the LCx. Ischemia was confirmed by immediate ECG changes. LV function was continuously recorded for 45 min. Contractility (LVdP/dtmax), relaxation (LVdP/dtmin) and end diastolic pressure (EDP) were less impaired in the betaARKct group as compared to PBS (P<0.05, two-way ANOVA). betaAR density was higher in the ischemic area of the LV in the betaARKct group (betaARKct: 71.9+/-4.6 fmol/mg protein, PBS: 54.5+/-4.0 fmol/mg protein, P<0.05). Adenylyl cyclase activity was also improved basally and in response to betaAR stimulation. betaARK1 activation was less in the betaARKct group (P<0.05). Therefore, inhibition of myocardial betaARK1 may represent a new strategy to prevent LV dysfunction induced by acute coronary ischemia.     

Structure meets function: actin filaments and myosin motors in the axon

This review focuses on recent advances in the understanding of the organization and roles of actin filaments, and associated myosin motor proteins, in regulating the structure and function of the axon shaft. ‘Patches’ of actin filaments have emerged as a major type of actin filament organization in axons. In the distal axon, patches function as precursors to the formation of filopodia and branches. At the axon initial segment, patches locally capture membranous organelles and contribute to polarized trafficking. The trapping function of patches at the initial segment can be ascribed to interactions with myosin motors, and likely also applies to patches in the more distal axon. Finally, submembranous rings of actin filaments were recently described in axons, which form an actin‐spectrin cytoskeleton, likely contributing to the maintenance of axon integrity. Continued investigation into the roles of axonal actin filaments and myosins will shed light on fundamental aspects of the development, adult function and the repair of axons in the nervous system.

     

Agrin triggers the clustering of raft-associated acetylcholine receptors through actin cytoskeleton reorganization

Background information. Cholesterol/sphingolipid‐rich membrane microdomains or membrane rafts have been implicated in various aspects of receptor function such as activation, trafficking and synapse localization. More specifically in muscle, membrane rafts are involved in AChR (acetylcholine receptor) clustering triggered by the neural factor agrin, a mechanism considered integral to NMJ (neuromuscular junction) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibres. Since membrane rafts are platforms sustaining actin nucleation, we hypothesize that these microdomains provide the suitable microenvironment favouring agrin/MuSK (mu scle‐s pecific k inase) signalling, eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signalling pathways operating through these microdomains still remains unclear. Results. In this work, we attempted to identify the interactions between membrane raft components and cortical skeleton that regulate, upon signalling by agrin, the assembly and stabilization of synaptic proteins of the postsynaptic membrane domain at the NMJ. We provide evidence that in C2C12 myotubes, agrin triggers the association of a subset of membrane rafts enriched in AChR, the ‐MuSK and Cdc42 (cell division cycle 42) to the actin cytoskeleton. Disruption of the liquid‐ordered phase by methyl‐β‐cyclodextrin abolished this association. We further show that actin and the actin‐nucleation factors, N‐WASP (neuronal Wiscott—Aldrich syndrome protein) and Arp2/3 (actin‐related protein 2/3) are transiently associated with rafts on agrin engagement. Consistent with these observations, pharmacological inhibition of N‐WASP activity perturbed agrin‐elicited AChR clustering. Finally, immunoelectron microscopic analyses of myotube membrane uncovered that AChRs were constitutively associated with raft nanodomains at steady state that progressively coalesced on agrin activation. These rearrangements of membrane domains correlated with the reorganization of cortical actin cytoskeleton through concomitant and transient recruitment of the Arp2/3 complex to AChR‐enriched rafts. Conclusions. The present observations support the notion that membrane rafts are involved in AChR clustering by promoting local actin cytoskeleton reorganization through the recruitment of effectors of the agrin/MuSK signalling cascade. These mechanisms are believed to play an important role in vivo in the formation of the NMJ.     

Interaction of plant polysomes with the actin cytoskeleton

Protein composition and functional activity of various polysome subpopulations isolated from Vicia faba L. leaves and Triticum aestivum L. and Hordeum vulgare L. seedlings were studied. Membrane- and cytoskeleton-bound polysomes were more active in the wheat germ cell-free translational system than free polysomes. Several non-ribosomal proteins were detected in the polysome preparations by gel electrophoresis and Western blot analysis: (1) a canonical actin of mol wt 42 kDa; (2) a 40 kDa protein, demonstrating affinity for ribosomes, sharing some determinants with actin, and present predominantly in the subpopulations of bound polysomes; and (3) an acidic ribosome-associated p40 evenly distributed between free and bound polysomes. The possibility of involvement of these proteins in interactions between polysomes and the actin cytoskeleton is discussed.     

The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

The B cell antigen receptor （BCR） is the sensor on the B cell surface that surveys foreign molecules （antigen） in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR- mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling.     

Three myosin V structures delineate essential features of chemo-mechanical transduction

The molecular motor, myosin, undergoes conformational changes in order to convert chemical energy into force production. Based on kinetic and structural considerations, we assert that three crystal forms of the myosin V motor delineate the conformational changes that myosin motors undergo upon detachment from actin. First, a motor domain structure demonstrates that nucleotide-free myosin V adopts a specific state (rigor-like) that is not influenced by crystal packing. A second structure reveals an actomyosin state that favors rapid release of ADP, and differs from the rigor-like state by a P-loop rearrangement. Comparison of these structures with a third structure, a 2.0 angstroms resolution structure of the motor bound to an ATP analog, illuminates the structural features that provide communication between the actin interface and nucleotide-binding site. Paramount among these is a region we name the transducer, which is composed of the seven-stranded beta-sheet and associated loops and linkers. Reminiscent of the beta-sheet distortion of the F1-ATPase, sequential distortion of this transducer region likely controls sequential release of products from the nucleotide pocket during force generation.     

Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton

Myosin VI, an actin-based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two-hybrid screens. In polarised epithelial cells, myosin VI is known to be associated with apical clathrin-coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low-density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP-2, whereas a C-terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalisation in clathrin-coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI–Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.     

A Role of myosin Vb and Rab11-FIP2 in the aquaporin-2 shuttle

Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.     

Moesin‐dependent cytoskeleton remodelling is associated with an anaplastic phenotype of pancreatic cancer

Cell motility is controlled by the dynamic cytoskeleton and its related proteins, such as members of the ezrin/radixin/moesin (ERM) family, which act as signalling molecules inducing cytoskeleton remodelling. Although ERM proteins have been identified as important factors in various malignancies, functional redundancy between these proteins has hindered the dissection of their individual contribution. The aim of the present study was to analyse the functional role of moesin in pancreatic malignancies. Cancer cells of different malignant lesions of human and transgenic mice pancreata were evaluated by immunohistochemistry. For functional analysis, cell growth, adhesion and invasion assays were carried out after transient and stable knock‐down of moesin expression in pancreatic cancer cells. In vivo tumourigenicity was determined using orthotopic and metastatic mouse tumour models. We now show that moesin knock‐down increases migration, invasion and metastasis and influences extracellular matrix organization of pancreatic cancer. Moesin‐regulated migratory activities of pancreatic cancer cells were in part promoted through cellular translocation of β‐catenin, and re‐distribution and organization of the cytoskeleton. Analysis of human and different transgenic mouse pancreatic cancers demonstrated that moesin is a phenotypic marker for anaplastic carcinoma, suggesting that this ERM protein plays a specific role in pancreatic carcinogenesis.     

Immunohistochemical detection of the retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) in the marbled newt along the annual cycle

Retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) expression in the testis of the marbled newt were investigated with special attention to the changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. The annual testicular cycle of the marbled newt (Triturus marmoratus marmoratus) comprises three periods: (a) proliferative period (germ cell proliferation from primordial germ cells to round spermatids, April-June); (b) spermiogenesis period (July-September); and (c) quiescence period (interstitial and follicular cells form the glandular tissue, October-April). In the proliferative period, primordial germ cells and primary spermatogonia immunostained intensely to the three types of RXRs and also to FXR. In the other periods, immunostaining to these antibodies was weak or absent. Secondary spermatogonia stained weakly to the four antibodies in the proliferative period, and only to FXR, also weakly, in the spermiogenesis period. Immunoreactive primary spermatocytes were weakly labeled with the RXR antibodies in the proliferative period. Spermatids and spermatozoa did not stain to any antibody in any period. Follicular cells only immunostained to RXR-gamma and only in the quiescence period when they are forming the glandular tissue, together with the interstitial cells. As follicular cells, interstitial cells only immunostained in the quiescence period; however, they immunoreacted to the three types of RXRs. These findings suggest that in the newt, RXRs and FXR are involved in spermatogenesis control by regulating the proliferation of primordial germ cells and spermatogonia. In addition, RXR-gamma seems to be also involved in the development of the glandular (steroidogenic) tissue.     

Rac1‐Rab11‐FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T‐cell activation

The immunological synapse generation and function is the result of a T‐cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11‐positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1‐dependent manner, key morphological events, like T‐cell spreading and synapse symmetry. Finally, Rab11‐/FIP3‐mediated regulation is necessary for T‐cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T‐cell activation.     

Splice-isoform specific immunolocalization of neuronal nitric oxide synthase in mouse and rat brain reveals that the PDZ-complex-building nNOSalpha beta-finger is largely exposed to antibodies

Knock out mice deficient for the splice-isoform alphaalpha of neuronal nitric oxide synthase (nNOSalphaalpha) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, betabeta and gammagamma, we generated isoform-specific anti-peptide antibodies against the nNOSalphaalpha specific betabeta-finger motif involved in PDZ domain scaffolding and the nNOSbetabeta specific N-terminus. The nNOSalphaalpha betabeta-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOSalphaalpha on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the betabeta-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOSalphaalpha betabeta-finger antibody in pull-down assays. By contrast, nNOSalphaalpha betabeta-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOSalphaalpha knock out mice, nNOSalphaalpha was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting betabeta/gammagamma-isoforms in these cells. The nNOSbetabeta antibody clearly detected bacterial expressed nNOSbetabeta fusion protein and nNOSbetabeta in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOSbetabeta in nNOSalphaalpha deficient animals.     

Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes

We have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick beta-actin mRNA and one oligonucleotide probe to chick alpha-cardiac actin mRNA. All the probes were 3' end-labeled with bio-11-dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin-alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of beta-actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of alpha-cardiac actin mRNA and the repression of beta-actin mRNA in differentiating myoblasts and in myotubes. With the alpha-cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When beta-actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of beta-actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. beta-Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both beta and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations.     

Regulation of the actin cytoskeleton by PIP2 in cytokinesis

Cytokinesis is a sequential process that occurs in three phases: assembly of the cytokinetic apparatus, furrow progression and fission (abscission) of the newly formed daughter cells. The ingression of the cleavage furrow is dependent on the constriction of an equatorial actomyosin ring in many cell types. Recent studies have demonstrated that this structure is highly dynamic and undergoes active polymerization and depolymerization throughout the furrowing process. Despite much progress in the identification of contractile ring components, little is known regarding the mechanism of its assembly and structural rearrangements. PIP2 (phosphatidylinositol 4,5-bisphosphate) is a critical regulator of actin dynamics and plays an essential role in cell motility and adhesion. Recent studies have indicated that an elevation of PIP2 at the cleavage furrow is a critical event for furrow stability. In this review we discuss the role of PIP2-mediated signalling in the structural maintenance of the contractile ring and furrow progression. In addition, we address the role of other phosphoinositides, PI(4)P (phosphatidylinositol 4-phosphate) and PIP3 (phosphatidylinositol 3,4,5-triphosphate) in these processes.     

Muscle actin isoforms are differentially expressed in human satellite cells isolated from donors of different ages

Myogenesis is mainly sustained by a subpopulation of myogenic cells known as satellite cells (SC). In this paper we studied alpha-smooth muscle (alphaSMA) and alpha-sarcomeric muscle (alphaSRA) actin isoform expression in cultures of human satellite cells (HSC) isolated from skeletal muscle biopsies from a 5-day-old newborn, a 34-year-old young adult and a 71-year-old donor. Myogenicity of cultures was assessed using immunocytochemical detection of desmin and myosin heavy chain. Time-course expression of alphaSRA and alphaSMA were studied with both immunocytochemistry and western blotting procedures. Although alphaSMA was never detected in whole skeletal muscle, both alphaSMA and alphaSRA were detected in proliferating and differentiating HSC derived from donors of all examined ages. The expression level experiments showed that alphaSRA was gradually up-regulated during HSC differentiation, but no significant differences were observed between newborn, young, and elderly HSC cultures. Our data demonstrated that HSC, isolated from subjects of different ages, re-expressed alphaSMA, but its levels and expression pattern varied considerably in the newborn with respect to the young adult and elderly donors. These results are discussed in relation to the myogenic differentiation capability of HSC during human muscle senescence.     

New methods for the isolation of skeletal muscle sarcolemma and sarcoplasmic reticulum allowing a comparison between the mammalian and amphibian beta(2)-adrenergic receptors and calcium pumps

New methods were established for the rapid and simultaneous isolation of multiple sarcolemmal and sarcoplasmic reticular fractions from very small amounts (0.25-2.0 g) of skeletal muscle. Thebeta(2)-adrenergic receptor and calcium transport systems were used as indices of purity and functional integrity as well as being the focal points of the study. These methods were found to be suitable for the special needs of small tissue samples, allowed rapid preparation and were appropriate for skeletal muscle from various species, frogs to mammals. The sarcolemmalbeta(2)-adrenergic receptor was expressed in frogs and mammals at similar levels of expression (336-454 fmol. x mg(-1)). The calcium pump was also present in sarcolemmal and sarcoplasmic reticular fractions in all species but notable species differences were found. In sarcolemmal fractions, while calcium binding was uniformly low (<1 nmol. x mg(-1)), oxalate stimulation was variable: low in frogs ( approximately 1.05-fold) high in mammals (120-450-fold). In sarcoplasmic reticular fractions, calcium binding was low in frogs (4-9 nmol. x mg(-1)) and much higher in mammals (322-383 nmol. x mg(-1)); oxalate stimulated calcium transport to a much greater extent in frogs (<70-fold) than in mammals (1.6-2-fold). It is concluded that thebeta(2)-adrenergic receptor appears to be strongly conserved in skeletal muscle while the use of calcium pumps evolves from reliance in Amphibia on the sarcoplasmic reticular calcium pump to the use in Mammalia of calcium pumps from both the sarcoplasmic reticulum and the plasma membrane.     

Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol

BACKGROUND: rac-Fenoterol is a beta2-adrenoceptor agonist (beta2-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R'- and S,S'-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the beta2-AR. METHODS: R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. Beta2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human beta2-AR and standard membrane binding studies using the same membranes. The effect of R-F and S-F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes. RESULTS: Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound beta2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 +/- 55 nM (R-F) and 109,000 +/- 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 +/- 11.6)% to (306 +/- 11.8)% of resting cell length (P < 0.05) and reduced EC50 from -7.0 +/- 0.270 to -7.1 +/- 0.2 log[M] (P < 0.05), while S-F had no significant effect. DISCUSSION: Previous studies have shown that rac-fenoterol acts as an apparent beta2-AR/G(s) selective agonist and fully restores diminished beta2-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for beta2-AR/G(s) activation and if it can be used in the treatment of congestive heart failure.