Confinement of β(1)- and β(2)-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β(1)- and β(2)AR, are structurally similar but mediate distinct signaling responses. Scaffold protein-mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β(1)- and β(2)AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)-domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β(2)AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β(2)AR confinement. For both β(1)- and β(2)AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β(1)- or β(2)AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes.     

The global spread of <Emphasis Type="Italic">Harmonia axyridis</Emphasis> (Coleoptera: Coccinellidae): distribution,dispersal and routes of invasion

Released as a biological control agent of aphids and coccids, Harmonia axyridis (Coleoptera: Coccinellidae) has spread from Asia to four additional continents. Since 1988 H. axyridis has established in at least 38 countries in its introduced range: three countries in North America, six in South America, 26 in Europe and three in Africa. In different continents the species has spread at rates estimated between 100 and 500 km year⁻¹. Here, the global spread of H. axyridis is thoroughly reviewed. Mechanisms of short- and long-distance dispersal in coccinellids are discussed, as are the reasons for them, with particular emphasis on H. axyridis. Dispersal via anthropogenic means has been particularly important in the case of H. axyridis. Preliminary studies investigating the invasion routes of H. axyridis using genetic analyses (involving both microsatellite and mitochondrial DNA) are outlined.     

Conserved regulation of p53 network dosage by microRNA-125b occurs through evolving miRNA-target gene pairs

MicroRNAs regulate networks of genes to orchestrate cellular functions. MiR-125b, the vertebrate homologue of the Caenorhabditis elegans microRNA lin-4, has been implicated in the regulation of neural and hematopoietic stem cell homeostasis, analogous to how lin-4 regulates stem cells in C. elegans. Depending on the cell context, miR-125b has been proposed to regulate both apoptosis and proliferation. Because the p53 network is a central regulator of both apoptosis and proliferation, the dual roles of miR-125b raise the question of what genes in the p53 network might be regulated by miR-125b. By using a gain- and loss-of-function screen for miR-125b targets in humans, mice, and zebrafish and by validating these targets with the luciferase assay and a novel miRNA pull-down assay, we demonstrate that miR-125b directly represses 20 novel targets in the p53 network. These targets include both apoptosis regulators like Bak1, Igfbp3, Itch, Puma, Prkra, Tp53inp1, Tp53, Zac1, and also cell-cycle regulators like cyclin C, Cdc25c, Cdkn2c, Edn1, Ppp1ca, Sel1l, in the p53 network. We found that, although each miRNA-target pair was seldom conserved, miR-125b regulation of the p53 pathway is conserved at the network level. Our results lead us to propose that miR-125b buffers and fine-tunes p53 network activity by regulating the dose of both proliferative and apoptotic regulators, with implications for tissue stem cell homeostasis and oncogenesis.     

Coupling of oligomerization and nucleotide binding in the AAA+ chaperone ClpB

Members of the family of ATPases associated with various cellular activities (AAA+) typically form homohexameric ring complexes and are able to remodel their substrates, such as misfolded proteins or protein-protein complexes, in an ATP-driven process. The molecular mechanism by which ATP hydrolysis is coordinated within the multimeric complex and the energy is converted into molecular motions, however, is poorly understood. This is partly due to the fact that the oligomers formed by AAA+ proteins represent a highly complex system and analysis depends on simplification and prior knowledge. Here, we present nucleotide binding and oligomer assembly kinetics of the AAA+ protein ClpB, a molecular chaperone that is able to disaggregate protein aggregates in concert with the DnaK chaperone system. ClpB bears two AAA+ domains (NBD1 and NBD2) on one subunit and forms homohexameric ring complexes. In order to dissect individual mechanistic steps, we made use of a reconstituted system based on two individual constructs bearing either the N-terminal (NBD1) or the C-terminal AAA+ domain (NBD2). In contrast to the C-terminal construct, the N-terminal construct does not bind the fluorescent nucleotide MANT-dADP in isolation. However, sequential mixing experiments suggest that NBD1 obtains nucleotide binding competence when incorporated into an oligomeric complex. These findings support a model in which nucleotide binding to NBD1 is dependent on and regulated by trans-acting elements from neighboring subunits, either by direct interaction with the nucleotide or by stabilization of a nucleotide binding-competent state. In this way, they provide a basis for intersubunit communication within the functional ClpB complex.     

Molecular pathways for intracellular cholesterol accumulation: common pathogenic mechanisms in Niemann-Pick disease Type C and cystic fibrosis

It has been less than two decades since the underlying genetic defects in Niemann-Pick disease Type C were first identified. These defects impair function of two proteins with a direct role in lipid trafficking, resulting in deposition of free cholesterol within late endosomal compartments and a multitude of effects on cell function and clinical manifestations. The rapid pace of research in this area has vastly improved our overall understanding of intracellular cholesterol homeostasis. Excessive cholesterol buildup has also been implicated in clinical manifestations associated with a number of genetically unrelated diseases including cystic fibrosis. Applying knowledge about anomalous cell signaling behavior in cystic fibrosis opens prospects for identifying similar previously unrecognized disease pathways in Niemann-Pick disease Type C. Recognition that Niemann-Pick disease Type C and cystic fibrosis both impair cholesterol regulatory pathways also provides a rationale for identifying common therapeutic targets.     

No Evidence for Prolonged Visible Persistence in Patients with Schizophrenia

Background

Temporal visual processing is strongly deteriorated in patients with schizophrenia. For example, the interval required between a visual stimulus and a subsequent mask has to be much longer in schizophrenic patients than in healthy controls. We investigated whether this deficit in temporal resolution is accompanied by prolonged visual persistence and/or deficient temporal precision (temporal asynchrony perception).

Methodology/Principal Findings

We investigated visual persistence in three experiments. In the first, measuring temporal processing by so-called backward masking, prolonged visible persistence is supposed to decrease performance. In the second experiment, requiring temporal integration, prolonged persistence is supposed to improve performance. In the third experiment, we investigated asynchrony detection, as another measure of temporal resolution. Eighteen patients with schizophrenia and 15 healthy controls participated. Asynchrony detection was intact in the patients. However, patients'' performance was inferior compared to healthy controls in the first two experiments. Hence, temporal processing in schizophrenic patients is indeed significantly impaired but this impairment is not caused by prolonged temporal integration.

Conclusions/Significance

Our results argue against a generally prolonged visual persistence in patients with schizophrenia. Together with the preserved ability of patients, to detect temporal asynchronies in permanently presented stimuli, the results indicate a more specific deficit in temporal processing of schizophrenic patients.     

Ultra Rapid Object Categorization: Effects of Level,Animacy and Context

It is widely agreed that in object categorization bottom-up and top-down influences interact. How top-down processes affect categorization has been primarily investigated in isolation, with only one higher level process at a time being manipulated. Here, we investigate the combination of different top-down influences (by varying the level of category, the animacy and the background of the object) and their effect on rapid object categorization. Subjects participated in a two-alternative forced choice rapid categorization task, while we measured accuracy and reaction times. Subjects had to categorize objects on the superordinate, basic or subordinate level. Objects belonged to the category animal or vehicle and each object was presented on a gray, congruent (upright) or incongruent (inverted) background. The results show that each top-down manipulation impacts object categorization and that they interact strongly. The best categorization was achieved on the superordinate level, providing no advantage for basic level in rapid categorization. Categorization between vehicles was faster than between animals on the basic level and vice versa on the subordinate level. Objects in homogenous gray background (context) yielded better overall performance than objects embedded in complex scenes, an effect most prominent on the subordinate level. An inverted background had no negative effect on object categorization compared to upright scenes. These results show how different top-down manipulations, such as category level, category type and background information, are related. We discuss the implications of top-down interactions on the interpretation of categorization results.