Acceptance and Commitment Therapy Group Intervention for Parents of Children with Disabilities (Navigator ACT): An Open Feasibility Trial.

Parents of children with autism spectrum disorder and other disabilities report high levels of distress, but systematically evaluated interventions are few. This study aimed to evaluate the feasibility of a novel, manualized Acceptance and Commitment Therapy group intervention (Navigator ACT) in a sample of 94 parents of children with disabilities. Feasibility was measured by treatment completion, credibility, and satisfaction, and preliminary outcomes by using self-rating scales administered at the baseline, post-intervention, and follow-up. The results imply the intervention is feasible in the context of Swedish outpatient habilitation services. A preliminary analysis of the outcome measures suggests that parents experienced significant improvements in well-being. The results indicate that the treatment is feasible and should be evaluated in a randomized controlled trial.

Magis - A magical adventure: Using a mobile game to deliver an ACT intervention for elementary schoolchildren in classroom settings.

Studies of the effects of the COVID-19 pandemic have shown that this health emergency has affected especially young people. Supporting the well-being of children is thus particularly urgent. However, the high prevalence of ill-being among children requires novel approaches to providing help. Health care resources are limited, and many children did not receive support even before the pandemic. The current study presents a novel approach to delivering brief interventions for school-aged children. A mobile game based on acceptance and commitment therapy was used to increase psychological flexibility and well-being among 10 to 12-year-old schoolchildren. A sample of 106 students played the game in four weekly sessions as part of normal teaching practice in school. The effectiveness of the brief game intervention was examined as a universal intervention among the whole sample and among subgroups created on the basis of baseline psychological flexibility (i.e., based on the need for an intervention). The results show that higher psychological flexibility was associated with less emotional and behavioral problems, higher health-related quality of life, mood, and school satisfaction, and less loneliness (r = 0.46-0.63). While a significant effect was not detected in the whole sample, the subsample of children with initially high psychological inflexibility benefitted from participating in the intervention (Cohen's d = 0.35). These preliminary findings suggest that the brief game-based intervention can increase psychological flexibility among children when the need for an intervention is considered. Further research is necessary to examine the stability of improvements in psychological flexibility.

Mechanisms of actin stress fibre assembly.

Stress fibres are contractile acto-myosin structures found from many types of non-muscle cells, where they are involved in adhesion, motility and morphogenesis. Stress fibres typically display a periodic alpha-actinin-myosin II pattern and are thus suggested to resemble the sarcomeric actin filament structures of muscle cells. Mammalian cells contain three categories of stress fibres: ventral stress fibres that are attached to focal adhesions at both ends, dorsal stress fibres that are attached to focal adhesions typically at one end and transverse arcs that are curved acto-myosin bundles, which do not directly attach to focal adhesions. In this review, we discuss the definition of stress fibres, organization of actin filaments and other components within these contractile structures, and the mechanisms of stress fibre assembly.

Regulation of the actin cytoskeleton by PI(4,5)P2 and PI(3,4,5)P3.

The actin cytoskeleton is fundamental for various motile and morphogenetic processes in cells. The structure and dynamics of the actin cytoskeleton are regulated by a wide array of actin-binding proteins, whose activities are controlled by various signal transduction pathways. Recent studies have shown that certain membrane phospholipids, especially PI(4,5)P2 and PI(3,4,5)P3, regulate actin filament assembly in cells and in cell extracts. PI(4,5)P2 appears to be a general regulator of actin polymerization at the plasma membrane or at membrane microdomains, whereas PI(3,4,5)P3 promotes the assembly of specialized actin filament structures in response to some growth factors. Biochemical studies have demonstrated that the activities of many proteins promoting actin assembly are upregulated by PI(4,5)P2, whereas proteins that inhibit actin assembly or promote filament disassembly are down-regulated by PI(4,5)P2. PI(3,4,5)P3 promotes its effects on the actin cytoskeleton mainly through activation of the Rho family of small GTPases. In addition to their effects on actin dynamics, both PI(4,5)P2 and PI(3,4,5)P3 promote the formation of specific actin filament structures through activation/inactivation of actin filament cross-linking proteins and proteins that mediate cytoskeleton-plasma membrane interactions.

Identification of yeast cofilin residues specific for actin monomer and PIP2 binding.

Cofilin/ADF is a ubiquitous actin-binding protein that is important for rapid actin dynamics in vivo. The long alpha-helix (helix 3 in yeast cofilin) forms the most highly conserved region in cofilin/ADF proteins, and residues in the NH2-terminal half of this alpha-helix have been shown to be essential for actin binding in cofilin/ADF. Recent studies also suggested that the basic residues in the COOH-terminal half of this alpha-helix would play an important role in F-actin binding. In contrast to these studies, we show here that the charged residues in the COOH-terminal half of helix 3 are not important for actin filament binding in yeast cofilin. Mutations in these residues, however, result in a small defect in actin monomer interactions. We also show that yeast cofilin can differentiate between various phosphatidylinositides, and mapped the PI(4,5)P2 binding site by using a collection of cofilin mutants. The PI(4,5)P2 binding site of yeast cofilin is a large positively charged surface that consists of residues in helix 3 as well as residues in other parts of the cofilin molecule. This suggests that cofilin/ADF proteins probably interact simultaneously with more than one PI(4,5)P2 molecule. The PI(4,5)P2-binding site overlaps with areas that are important for F-actin binding, explaining why the actin-related activities of cofilin/ADF are inhibited by PI(4,5)P2. The biological roles of actin and PI(4,5)P2 interactions of cofilin are discussed in light of phenotypes of specific yeast strains carrying mutations in residues that are important for actin and PI(4,5)P2 binding.

Twinfilin is required for actin-dependent developmental processes in Drosophila.

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer-binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes.

Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin.

Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics.

In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin. Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase. In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein. However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro. A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen. In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures. A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/twinfilin localization to cytoplasm. Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.

Aip1p interacts with cofilin to disassemble actin filaments.

Actin interacting protein 1 (Aip1) is a conserved component of the actin cytoskeleton first identified in a two-hybrid screen against yeast actin. Here, we report that Aip1p also interacts with the ubiquitous actin depolymerizing factor cofilin. A two-hybrid-based approach using cofilin and actin mutants identified residues necessary for the interaction of actin, cofilin, and Aip1p in an apparent ternary complex. Deletion of the AIP1 gene is lethal in combination with cofilin mutants or act1-159, an actin mutation that slows the rate of actin filament disassembly in vivo. Aip1p localizes to cortical actin patches in yeast cells, and this localization is disrupted by specific actin and cofilin mutations. Further, Aip1p is required to restrict cofilin localization to cortical patches. Finally, biochemical analyses show that Aip1p causes net depolymerization of actin filaments only in the presence of cofilin and that cofilin enhances binding of Aip1p to actin filaments. We conclude that Aip1p is a cofilin-associated protein that enhances the filament disassembly activity of cofilin and restricts cofilin localization to cortical actin patches.

Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein.

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)-twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.

Essential functions and actin-binding surfaces of yeast cofilin revealed by systematic mutagenesis.

Cofilin stimulates actin filament turnover in vivo. The phenotypes of twenty yeast cofilin mutants generated by systematic mutagenesis were determined. Ten grew as well as the wild type and showed no cytoskeleton defects, seven were recessive-lethal and three were conditional-lethal and caused severe actin organization defects. Biochemical characterization of interactions between nine mutant yeast cofilins and yeast actin provided evidence that F-actin binding and depolymerization are essential cofilin functions. Locating the mutated residues on the yeast cofilin molecular structure allowed several important conclusions to be drawn. First, residues required for actin monomer binding are proximal to each other. Secondly, additional residues are required for interactions with actin filaments; these residues might bind an adjacent subunit in the actin filament. Thirdly, despite striking structural similarity, cofilin interacts with actin in a different manner from gelsolin segment-1. Fourthly, a previously unrecognized cofilin function or interaction is suggested by identification of spatially proximal residues important for cofilin function in vivo, but not for actin interactions in vitro. Finally, mutation of the cofilin N-terminus suggests that its sequence is conserved because of its critical role in actin interactions, not because it is sometimes a target for protein kinases.

Cofilin promotes rapid actin filament turnover in vivo.

The ability of actin filaments to function in cell morphogenesis and motility is coupled to their capacity for rapid assembly and disassembly. Because disassembly in vitro is much slower than in vivo, cellular factors that stimulate disassembly have long been assumed to exist. Although numerous proteins can affect actin dynamics in vitro, demonstration of in vivo relevance of these effects has not been achieved. We have used genetics and an actin-inhibitor in yeast to demonstrate that rapid cycles of actin assembly and disassembly depend on the small actin-binding protein cofilin, and that cofilin stimulates filament disassembly. These results may explain why cofilin is ubiquitous in eukaryotes and is essential for viability in every organism in which its function has been tested genetically. Magnitudes of disassembly defects in cofilin mutants in vivo were found to be correlated closely with the magnitudes of disassembly defects observed in vitro, supporting our conclusions. Furthermore, these cofilin mutants provided an opportunity to distinguish in living cells those actin functions that depend specifically on filament turnover (endocytosis) from those that do not (cortical actin patch motility).

Structure determination of yeast cofilin.

Cofilin, a ubiquitous 15,000 M(r) protein, plays a central role in regulating cytoskeletal dynamics. Cofilin binds to actin monomers and filaments, and has a pH-dependent actin severing activity. The structure will allow for a detailed analysis of cofilin function.

Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center.

Cytochrome oxidase is a membrane protein complex that catalyzes reduction of molecular oxygen to water and utilizes the free energy of this reaction to generate a transmembrane proton gradient during respiration. The electron entry site in subunit II is a mixed-valence dinuclear copper center in enzymes that oxidize cytochrome c. This center has been lost during the evolution of the quinoloxidizing branch of cytochrome oxidases but can be restored by engineering. Herein we describe the crystal structures of the periplasmic fragment from the wild-type subunit II (CyoA) of Escherichia coli quinol oxidase at 2.5-A resolution and of the mutant with the engineered dinuclear copper center (purple CyoA) at 2.3-A resolution. CyoA is folded as an 11-stranded mostly antiparallel beta-sandwich followed by three alpha-helices. The dinuclear copper center is located at the loops between strands beta 5-beta 6 and beta 9-beta 10. The two coppers are at a 2.5-A distance and symmetrically coordinated to the main ligands that are two bridging cysteines and two terminal histidines. The residues that are distinct in cytochrome c and quinol oxidases are around the dinuclear copper center. Structural comparison suggests a common ancestry for subunit II of cytochrome oxidase and blue copper-binding proteins.

Spectroscopic and mutagenesis studies on the CuA centre from the cytochrome-c oxidase complex of Paracoccus denitrificans.

Cytochrome-c oxidase contains an unusual copper centre (CuA) located in subunit II. This centre mediates one-electron transfer from cytochrome c to low-spin heme a. Recent spectroscopic and biochemical studies have shown that this centre is a valence delocalised dinuclear [Cu(+1.5)-Cu(+1.5)] centre. We have measured the absorption, EPR and variable-temperature magnetic circular dichroism spectra of the CuA-binding domain isolated from Paracoccus denitrificans cytochrome aa3. The EPR spectrum showed the following signals: gparallel = 2.18; gperpendicular = 2.03. gparallel exhibited a seven-line hyperfine splitting pattern, with an intensity ratio showing that the single unpaired electron interacted equally with two copper nuclei. The magnetic circular dichroism spectrum was identical to those from CuA in bovine heart cytochrome-c oxidase and centre A of nitrous-oxide reductase, showing the close structural similarity between the three centres. To identify the ligands of CuA, all the conserved putative ligands in the P. denitrificans CuA domain were substituted. Only five residues, Cys244, Cys248, His209, His252, and Met255, were required for correct assembly of the CuA centre. Replacement of Met255 caused protein misfolding. Hence, methionine may have a structural role for the folding of the protein rather than being a CuA ligand. Given that both copper ions must have identical coordination geometries, the number of possible structures is limited. Two models are proposed: one involves the thiolate side-chains of Cys244 and Cys248 bridging a pair of copper ions with one histidine coordinating each copper ion, and the other has terminal ligation of each copper ion by one cysteine and one histidine residue. In both models, the metal-metal distance can be sufficiently short to permit direct d-orbital overlap of the copper ions. The magnetic circular dichroism transitions at 475 nm and 525 nm are assigned to thiolate-to-copper charge-transfer processes polarised perpendicular to one another, although the magnetic circular dichroism intensities show that the excited states were heavily mixed with copper d-orbitals. These intensities can be interpreted in the thiolate bridged model in terms of transitions within a Cu2(SR)2 rhomb. In the model involving terminal cysteine ligation, exciton coupling of two thiolate-to-copper charge-transfer transitions of similar energy, polarised along the Cu-S bonds, would contribute two transitions perpendicular to one another. This requires that the cysteine ligands have a cis orientation relative to one another.(ABSTRACT TRUNCATED AT 400 WORDS)

Electron transfer between cytochrome c and the isolated CuA domain: identification of substrate-binding residues in cytochrome c oxidase.

Subunit II of cytochrome c oxidase has a C-terminal domain that is exposed to aqueous solution on membrane surface and contains a copper center called CuA. The central part of the cytochrome c binding site is thought to reside in this domain. We have expressed the subunit II fragment of the Paracoccus denitrificans cytochrome c oxidase in a soluble form and studied its interaction with cytochrome c by stopped-flow spectroscopy. The oxidation of cytochrome c by the CuA domain follows monophasic kinetics, indicating the presence of a single kinetically competent binding site. In low ionic strength medium, the domain oxidizes Paracoccus cytochrome c-550 and horse mitochondrial cytochrome c at the rates of 1.5 x 10(6) and 3 x 10(5) M-1 s-1, respectively. The reaction rates are strongly dependent on ionic strength, which must reflect electrostatic interactions within the complex. The KD for the complex between the bacterial cytochrome c and the domain is 1.6 microM; i.e., it is similar to that between the mitochondrial cytochrome c and the intact oxidase, suggesting that both contain the same catalytically competent binding site. Using site-directed mutagenesis, we have identified five conserved residues of the CuA domain that are involved in the cytochrome c binding. Mutations of glutamine 148, glutamate 154, aspartate 206, aspartate 221, or glutamate 246 lead to a 35-85% decrease in the rate of cytochrome c oxidation. The simultaneous substitution of three invariant carboxylic acids (aspartate 206, aspartate 221, and glutamate 246) leads to a 95% decrease in the reaction rate. Conversely, the reaction can be enhanced by removing a positive charge (lysine 219) from the CuA domain.

Detection of mosquito saliva-specific IgE and IgG4 antibodies by immunoblotting.

IgE and IgG subclass antibodies against Aedes communis mosquito saliva were studied by immunoblotting in 12 adults with immediate and/or delayed skin reactions to mosquito bites. Four antigenic proteins, with molecular weights of 22, 30, 36, and 64 kd, were found in the mosquito saliva. Almost all subjects (11 of 12) had anti-mosquito saliva-specific IgE antibodies directed against the 36 kd protein. The IgG antibody response appeared to be restricted mostly to IgG4 (11 of 12) and IgG1 (8 of 11) subclasses against the same 36 kd antigen. Ten of the 12 subjects had both IgE and IgG4 antibodies to the 36 kd protein. No anti-mosquito antibodies were found in pooled sera of five infants never exposed to mosquito bites. These results show that most persons with immediate skin reactivity to A. communis mosquito bites have both IgE and IgG4 antibodies that recognize the 36 kd antigen present in the mosquito saliva, suggesting that anti-saliva antibodies may play a role in the pathogenesis of mosquito bite reactions.

Soluble CuA-binding domain from the Paracoccus cytochrome c oxidase.

In cytochrome c oxidase the C-terminal part of subunit II is outside the membrane and contains a copper center called CuA. We have expressed this domain of the Paracoccus denitrificans oxidase in a soluble form. Data obtained by quantitative copper-to-protein measurements, electrospray mass spectrometry, and electron paramagnetic resonance spectroscopy show that the center contains two copper atoms probably in a mixed valence configuration. Its absorbance spectrum is similar to that of the copper center A in nitrous oxide reductase. The EPR spectrum suggests that the center in the soluble protein is closely related to the native CuA site in the cytochrome oxidase complex. However, it seems likely that the copper center in the soluble domain is more exposed to the aqueous milieu than in the intact complex because its absorbance and EPR spectra are sensitive to pH. At alkaline pH one of the coppers in the site acquires type-2 character, indicating that it may be coordinated to a new ligand. The pK of this reversible change is about 8.2. The CuA-binding fragment is able to oxidize cytochrome c.

Two cysteines, two histidines, and one methionine are ligands of a binuclear purple copper center.

Cytochrome c oxidase contains a copper center, CuA, which is involved in electron transfer from cytochrome c to the oxygen-reducing active site. This center is distinct from types 1, 2, and 3 copper sites and related only to a purple copper center in nitrous oxide reductase. At present it is not clear whether this site is mononuclear or is comprised of two copper atoms in a mixed valence (Cu(I)-Cu(II)) configuration. Here we use a model of CuA, engineered into a structurally related but initially copperless protein, to study the structure of this copper center. The results from biochemical analysis, site-directed mutagenesis, and electrospray mass spectrometry support the binuclear model. Two cysteines, two histidines, and one methionine are the major ligands of two coppers. Substitution of these residues results in either a complete loss of color or dramatic changes in the absorbance spectrum. In contrast, substitution of the invariant glutamate residue, which is located between the copper-binding cysteines, leads to a minor perturbation of the optical spectrum.