Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5-aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity

In archaeal rRNAs, the isomerization of uridine into pseudouridine (Ψ) is achieved by the H/ACA sRNPs and the minimal set of proteins required for RNA:Ψ-synthase activity is the aCBF5–aNOP10 protein pair. The crystal structure of the aCBF5–aNOP10 heterodimer from Pyrococcus abyssi was solved at 2.1 Å resolution. In this structure, protein aNOP10 has an extended shape, with a zinc-binding motif at the N-terminus and an α-helix at the C-terminus. Both motifs contact the aCBF5 catalytic domain. Although less efficiently as does the full-length aNOP10, the aNOP10 C-terminal domain binds aCBF5 and stimulates the RNA-guided activity. We show that the C-terminal domain of aCBF5 (the PUA domain), which is wrapped by an N-terminal extension of aCBF5, plays a crucial role for aCBF5 binding to the guide sRNA. Addition of this domain in trans partially complement particles assembled with an aCBF5ΔPUA truncated protein. In the crystal structure, the aCBF5–aNOP10 complex forms two kinds of heterotetramers with parallel and perpendicular orientations of the aNOP10 terminal α-helices, respectively. By gel filtration assay, we showed that aNOP10 can dimerize in solution. As both residues Y41 and L48 were needed for dimerization, the dimerization likely takes place by interaction of parallel α-helices.     

Defective transfer of seminal-fluid materials during matings of semi-fertile fruitless mutants in Drosophila

In context of the semi-sterility exhibited by Drosophila males expressing certain mating-enabling fruitless (fru) mutant genotypes, we examined the transfer of seminal fluid using a transgene that encodes the Sex Peptide (SP) oligopeptide fused to Green Fluorescent Protein (GFP). We found that this fusion construct expresses SP-GFP in a valid manner within accessory glands of the male reproductive system in normal and fru-mutant males. Transfer of SP-GFP to live females was readily detectable during and after copulation. With respect to the pertinent combinations of fru mutations, we demonstrated that these abnormal genotypes cause males to transmit mating-related materials in two aberrant ways: one involving whether any seminal-fluid entities are transferred at all during a given mating; the other revealing an intriguing aspect of these fruitless effects, such that the mutations in question cause males to transfer female-affecting materials in a manner that varies among copulations. In this regard, certain mutant males that do not transfer SP nevertheless are able to transfer sperm: a fru-mated female possessing no GFP who was not fecund initially could produce progeny when seminal-fluid proteins were subsequently supplied by mating with a male that was spermless owing to the effects of a tudor mutation.     

Positioning centrosomes and spindle poles: looking at the periphery to find the centre

Centrosome positioning is tightly controlled throughout the cell cycle and probably shares common regulatory mechanisms with spindle-pole positioning. In this article, we detail the possible mechanisms controlling centrosome and spindle positioning in various organisms both in interphase and mitotic cells, and discuss recent findings showing how microtubule plus-end-associated proteins interact with the cell cortex. We suggest that microtubule plus-end complexes simultaneously regulate microtubule dynamics and microtubule anchoring at the cell periphery to allow proper centrosome and spindle-pole positioning.     

The evolution of mutation rate in finite asexual populations

In this article, we model analytically the evolution of mutation rate in asexual organisms. Three selective forces are present. First, everything else being equal, individuals with higher mutation rate have a larger fitness, thanks to the energy and time saved by not replicating DNA accurately. Second, as a flip side, the genome of these individuals is replicated with errors that may negatively affect fitness. Third, and conversely, replication errors have a potential benefit if beneficial mutations are to be generated. Our model describes the fate of modifiers of mutation rate under the three forces and allows us to predict the long-term evolutionary trajectory of mutation rate. We obtain three major results. First, in asexuals, the needs for both adaptation and genome preservation are not evolutionary forces that can stabilize mutation rate at an intermediate optimum. When adaptation has a significant role, it primarily destabilizes mutation rate and yields the emergence of strong-effect mutators. Second, in contrast to what is usually believed, the appearance of modifiers with large mutation rate is more likely when the fitness cost of each deleterious mutation is weak, because the cost of replication errors is then paid after a delay. Third, in small populations, and even if adaptations are needed, mutation rate is always blocked at the minimum attainable level, because the rate of adaptation is too slow to play a significant role. Only populations whose size is above a critical mass see their mutation rate affected by the need for adaptation.     

Observing the confinement potential of bacterial pore-forming toxin receptors inside rafts with nonblinking Eu(3+)-doped oxide nanoparticles

We track single toxin receptors on the apical cell membrane of MDCK cells with Eu-doped oxide nanoparticles coupled to two toxins of the pore-forming toxin family: α-toxin of Clostridium septicum and ε-toxin of Clostridium perfringens. These nonblinking and photostable labels do not perturb the motion of the toxin receptors and yield long uninterrupted trajectories with mean localization precision of 30 nm for acquisition times of 51.3 ms. We were thus able to study the toxin-cell interaction at the single-molecule level. Toxins bind to receptors that are confined within zones of mean area 0.40 ± 0.05 μm(2). Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 ± 0.01 μm(2)/s for both toxin receptors. Moreover, application of this approach revealed a force field within the domain generated by a springlike confining potential. Both toxin receptors were found to experience forces characterized by a mean spring constant of 0.30 ± 0.03 pN/μm at 37°C. Furthermore, both toxin receptors showed similar distributions of diffusion coefficient, domain area, and spring constant. Control experiments before and after incubation with cholesterol oxidase and sphingomyelinase show that these two enzymes disrupt the confinement domains and lead to quasi-free motion of the toxin receptors. Our control data showing cholesterol and sphingomyelin dependence as well as independence of actin depolymerization and microtubule disruption lead us to attribute the confinement of both receptors to lipid rafts. These toxins require oligomerization to develop their toxic activity. The confined nature of the toxin receptors leads to a local enhancement of the toxin monomer concentration and may thus explain the virulence of this toxin family.     

The C-terminal polyproline-containing region of ELMO contributes to an increase in the life-time of the ELMO-DOCK complex

The eukaryotic Engulfment and CellMotility (ELMO) proteins form an evolutionary conserved family of key regulators which play a central role in Rho-dependent biological processes such as engulfment and cell motility/migration. ELMO proteins interact with a subset of Downstream of Crk (DOCK) family members, a new type of guanine exchange factors (GEF) for Rac and cdc42 GTPases. The physiological function of DOCK is to facilitate actin remodeling, a process which occurs only in presence of ELMO. Several studies have determined that the last 200 C-terminal residues of ELMO1 and the first 180 N-terminal residues of DOCK180 are responsible for the ELMO-DOCK interaction. However, the precise role of the different domains and motifs identified in these regions has remained elusive. Divergent functional, biochemical and structural data have been reported regarding the contribution of the C-terminal end of ELMO, comprising its polyproline motif, and of the DOCK SH3 domain. In the present study, we have investigated the contribution of the C-terminal end of ELMO1 to the interaction between ELMO1 and the SH3 domain of DOCK180 using nuclear magnetic resonance spectroscopy and surface plasmon resonance. Our data presented here demonstrate the ability of the SH3 domain of DOCK180 to interact with ELMO1, regardless of the presence of the polyproline-containing C-terminal end. However, the presence of the polyproline region leads to a significant increase in the half-life of the ELMO1-DOCK180 complex, along with a moderate increase on the affinity.     

Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.     

Phenotypic plasticity influences the size, shape and dynamics of the geographic distribution of an invasive plant

Phenotypic plasticity has long been suspected to allow invasive species to expand their geographic range across large-scale environmental gradients. We tested this possibility in Australia using a continental scale survey of the invasive tree Parkinsonia aculeata (Fabaceae) in twenty-three sites distributed across four climate regions and three habitat types. Using tree-level responses, we detected a trade-off between seed mass and seed number across the moisture gradient. Individual trees plastically and reversibly produced many small seeds at dry sites or years, and few big seeds at wet sites and years. Bigger seeds were positively correlated with higher seed and seedling survival rates. The trade-off, the relation between seed mass, seed and seedling survival, and other fitness components of the plant life-cycle were integrated within a matrix population model. The model confirms that the plastic response resulted in average fitness benefits across the life-cycle. Plasticity resulted in average fitness being positively maintained at the wet and dry range margins where extinction risks would otherwise have been high ("Jack-of-all-Trades" strategy JT), and fitness being maximized at the species range centre where extinction risks were already low ("Master-of-Some" strategy MS). The resulting hybrid "Jack-and-Master" strategy (JM) broadened the geographic range and amplified average fitness in the range centre. Our study provides the first empirical evidence for a JM species. It also confirms mechanistically the importance of phenotypic plasticity in determining the size, the shape and the dynamic of a species distribution. The JM allows rapid and reversible phenotypic responses to new or changing moisture conditions at different scales, providing the species with definite advantages over genetic adaptation when invading diverse and variable environments. Furthermore, natural selection pressure acting on phenotypic plasticity is predicted to result in maintenance of the JT and strengthening of the MS, further enhancing the species invasiveness in its range centre.     

Belief revision and delusions: how do patients with schizophrenia take advice?

The dominant cognitive model that accounts for the persistence of delusional beliefs in schizophrenia postulates that patients suffer from a general deficit in belief revision. It is generally assumed that this deficit is a consequence of impaired reasoning skills. However, the possibility that such inflexibility affects the entire system of a patient's beliefs has rarely been empirically tested. Using delusion-neutral material in a well-documented advice-taking task, the present study reports that patients with schizophrenia: 1) revise their beliefs, 2) take into account socially provided information to do so, 3) are not overconfident about their judgments, and 4) show less egocentric advice-discounting than controls. This study thus shows that delusional patients' difficulty in revising beliefs is more selective than had been previously assumed. The specificities of the task and the implications for a theory of delusion formation are discussed.