Structural basis for endosomal targeting by the Bro1 domain

Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs.     

Endophilin B1/Bif-1 Stimulates BAX Activation Independently from Its Capacity to Produce Large Scale Membrane Morphological Rearrangements

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes. In this reconstituted model system the N-BAR domain of Bif-1 reproduced the stimulatory effect of Bif-1 on functional BAX activation. This process was dependent on physical interaction between Bif-1 N-BAR and BAX as well as on the presence of the mitochondrion-specific lipid cardiolipin. Despite that Bif-1 N-BAR produced large scale morphological rearrangements in MOM-like liposomes, this phenomenon could be separated from functional BAX activation. Furthermore, DLP1 also caused global morphological changes in MOM-like liposomes, but DLP1 did not stimulate BAX-permeabilizing function in the absence or presence of Bif-1. Taken together, our findings not only provide direct evidence for a functional interplay between Bif-1, BAX, and cardiolipin during MOMP but also add significantly to the growing body of evidence indicating that components of the mitochondrial morphogenesis machinery possess proapoptotic functions that are independent from their recognized roles in normal mitochondrial dynamics.MOMP³ is a key event in the intrinsic pathway of mammalian apoptosis, resulting in the release of several apoptogenic proteins from the mitochondrial intermembrane space into the cytosol (1). Released intraorganellar components, including cytochrome c, Smac/DIABLO, and AIF, then act as mediators for activating executioner caspase proteases or for other downstream events in the intracellular apoptosis cascade. MOMP is tightly regulated by BCL-2 family members, whose core components are proapoptotic BAX-type proteins that directly effect MOMP and antiapoptotic BCL-2-type proteins which inhibit MOMP (2, 3). In a currently popular model, a third subgroup of BCL-2 family proteins, the BH3-only proteins, trigger a set of conformational changes in BAX and/or its close homologue BAK that activates their permeabilizing function, thereby causing MOMP.Multiple proteins implicated in mitochondrial morphogenesis during normal growth conditions can cross-talk with BCL-2 family members to affect the mitochondrial pathway of apoptosis (4). For example, the large dynamin-like GTPase DLP1/Drp1 and hFis1, two essential components of the mitochondrial fission machinery, have been shown to modulate pro-apoptotic BAX function and mitochondrial cytochrome c release by acting at the level of the MOM (5–7). However, although excessive mitochondrial fragmentation is characteristic in mammalian apoptosis, controversy persists as to whether this phenomenon is merely coincident with or causatively linked to MOMP induction (4 – 8). In addition, a considerable body of evidence has amassed indicating that DLP1/Drp1 and hFis1 are multifunctional proteins that do not use the same mechanisms to reshape mitochondria in healthy conditions and to promote release of mitochondrial intermembrane space proteins during apoptosis (7–10).Endophilin B1/BAX-interacting factor 1 (Bif-1) is another protein linking mitochondrial morphological changes and BCL-2-regulated programmed cell death (4). On the one hand, Bif-1 is known to participate downstream of DLP1/Drp1, modulating normal MOM morphological dynamics in healthy cells (11). On the other hand, in response to specific apoptotic signals, a significant portion of Bif-1 binds BAX at the MOM in close temporal correlation with BAX conformational change and cytochrome c release (12). In addition, increasing the levels of Bif-1 has been shown to accelerate BAX conformational change, caspase activation, and apoptotic cell death, whereas loss of Bif-1 delays all these processes (12, 13). Together, these previous findings point to an important contributing role of Bif-1 in BAX-driven MOMP during apoptosis, but the underlying molecular mechanism remains unknown.As other members of the endophilin family, Bif-1 contains an N-BAR (Bin-amphiphysin-Rvs) domain that has been shown to confer ability to these proteins for transforming flat lipid bilayers into high curvature buds, tubules, and vesicles in vitro (14–17). Crystallographic studies of the N-BAR domain of endophilin A1, a close homologue of Bif-1, revealed a crescent-shape homodimer with a positively charged concave surface which is believed to act like a molecular scaffold that impresses its own curvature on binding to negatively charged membranes (16, 18). Another distinguishing feature of endophilin N-BAR domains is the presence of two distinct amphipathic segments referred to as “Helix 0” (H0) and “Helix 1 insert” (H1I) that penetrate only partway into the external leaflet and are thought to create a wedge effect that also increases membrane curvature. This dual curvature-generating mechanism has been linked to the shared capacity of endophilins to operate in membrane tubulovesicular dynamics during normal cell growth together with dynamin/dynamin-like proteins (16–18). However, exactly how the molecular-scale perturbation of membrane curvature induced by N-BAR domains translates into large scale membrane remodeling processes (e.g. tubulation and vesiculation) is not well understood (19–25). In addition, it is unclear whether the ability of Bif-1 to produce global changes in membrane morphology is functionally connected to its apoptotic mode of action.The complexity of the network of intermolecular interactions that controls the BCL-2-regulated MOMP pathway constitutes a major hurdle for gaining a molecular-level understanding of Bif-1 pro-death function in intact cells. Another complicating factor is that Bif-1 can interact with binding partners other than BAX at intracellular membranes distinct from the MOM depending on environmental conditions (14, 26–29). In previous studies this and other laboratories have shown that the BCL-2-regulated MOMP pathway can be reconstituted in a simplified system consisting of purified recombinant proteins and chemically defined MOM-like large unilamellar vesicles (LUV) in a manner that faithfully reflects the basic physiological functions of BCL-2 family proteins at the MOM (30–33). Here, we have used this minimal cell-free system to advance our understanding of the pro-death role of Bif-1. We provide strong evidence for a direct implication of Bif-1 in functional BAX activation at the membrane level and novel insights concerning the mechanism through which Bif-1 achieves this effect.     

Filling the North European Early/Middle Eocene (Ypresian/Lutetian) boundary gap: Insights from the Pyrenean continental to deep-marine record

The Early/Middle Eocene (Ypresian/Lutetian) transition is represented by a hiatus in many North European sections, including those in which the classic stratotypes were originally defined. However, the Global Stratotype Section and Point of the Lutetian Stage, which is still pending definition, should be placed at a globally correlatable event included within that unrepresented interval. The Pyrenean Eocene outcrops display sedimentary successions that offer the rare opportunity to analyse the Ypresian/Lutetian boundary interval in almost continuous sections and in very different settings. Seven reference stratigraphic sections were selected on the basis of their quality and correlated by means of biomagnetostratigraphic data. This correlation framework casts light on the sequence of chronostratigraphic events that characterize the Ypresian/Lutetian boundary interval, which may prove useful in defining the main correlation criterion of the base of the Lutetian.All of the Pyrenean sections show a similar sedimentary evolution, despite being up to 350 km apart from each other, containing deposits of different origins (intrabasinal carbonate sediments, siliciclastic sediments sourced from the Iberian plate, and terrigenous sediments sourced from the uplifting Pyrenees) and despite having been accumulated in different sedimentary environments (from continental to deep marine) and in different geodynamic settings (piggy-back basin, foreland basin and cratonic margin). This common evolution can be readily interpreted in terms of a sea-level driven depositional sequence whose lowstand and transgressive systems tracts are included within the Ypresian/Lutetian boundary interval. The Pyrenean Ypresian/Lutetian depositional sequence can reasonably be correlated with depositional sequences from classic North European areas, shedding light on the palaeoenvironmental history which in those areas has not been recorded. Furthermore, these depositional sequences may possibly correlate with others from the Antarctic Ocean and from New Jersey, as well as with oceanic temperature variations, suggesting that they might be the result of climatically-driven glacioeustatic sea-level changes. Should this hypothesis prove correct, it would confirm previous suggestions that the onset of Antarctic glaciations needs to be backshifted to the late Ypresian at least.     

Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis

The engineering of a full-length infectious cDNA clone and a functional replicon of the severe acute respiratory syndrome coronavirus (SARS-CoV) Urbani strain as bacterial artificial chromosomes (BACs) is described in this study. In this system, the viral RNA was expressed in the cell nucleus under the control of the cytomegalovirus promoter and further amplified in the cytoplasm by the viral replicase. Both the infectious clone and the replicon were fully stable in Escherichia coli. Using the SARS-CoV replicon, we have shown that the recently described RNA-processing enzymes exoribonuclease, endoribonuclease, and 2'-O-ribose methyltransferase were essential for efficient coronavirus RNA synthesis. The SARS reverse genetic system developed as a BAC constitutes a useful tool for the study of fundamental viral processes and also for developing genetically defined vaccines.     

DNA microarray technology: a new tool for the epidemiological typing of bacterial pathogens?

Genomic hybridization on whole genome arrays detects the presence or absence of similar DNA regions in sufficiently related microorganisms, allowing genome-wide comparison of their genetic contents. A whole genome array is based on a sequenced bacterial isolate, and is a collection of DNA probes fixed on a solid support. In a single hybridization experiment, the absence/presence status of all genes of the sequenced microbe in the queried isolate can be examined. The objective of this minireview is to summarize the past usage of DNA microarray technology for microbial strain characterizations, and to estimate its future utilization in epidemiological studies and molecular typing of bacterial pathogens. The studies reviewed here confirm the usefulness of microarray technology for the detection of genetic polymorphisms. However, the construction or purchase of DNA microarrays and the performance of strain to strain hybridization experiments are still prohibitively expensive for routine application. Future use of arrays in epidemiology is likely to depend on the development of more cost-effective protocols, more robust and simplified formats, and the adequate evaluation of their performance (efficacy) and convenience (efficiency) compared with other genotyping methods. It seems more likely that a more focused assay, concentrating on genomic regions of variability previously detected by genome-wide microarrays, will find broad application in routine bacterial epidemiology.     

Fecundity limitation of Calanus helgolandicus, by the parasite Ellobiopsis sp.

The effect of the parasite Ellobiopsis sp., on the fecundityof Calanus helgolandicus and Calanoides carinatus in the Bayof Biscay, was investigated in May 2003. An average of 6.8%of C. helgolandicus females were infected with Ellobiopsis sp.,whereas none of the C. carinatus were found to be infected.An objective method of estimating gonad development was appliedto quantitatively measure the effect of the parasitism on thereproduction of the copepod. Parasitism by Ellobiopsis sp. hasthe potential to reduce the fecundity of C. helgolandicus females.     

Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active “soluble AC”. The calpain-mediated ACT processing allows trafficking of the “soluble AC” domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP “pools”, which would play different roles in the cell pathophysiology.     

Bystander Responses to a Violent Incident in an Immersive Virtual Environment

Under what conditions will a bystander intervene to try to stop a violent attack by one person on another? It is generally believed that the greater the size of the crowd of bystanders, the less the chance that any of them will intervene. A complementary model is that social identity is critical as an explanatory variable. For example, when the bystander shares common social identity with the victim the probability of intervention is enhanced, other things being equal. However, it is generally not possible to study such hypotheses experimentally for practical and ethical reasons. Here we show that an experiment that depicts a violent incident at life-size in immersive virtual reality lends support to the social identity explanation. 40 male supporters of Arsenal Football Club in England were recruited for a two-factor between-groups experiment: the victim was either an Arsenal supporter or not (in-group/out-group), and looked towards the participant for help or not during the confrontation. The response variables were the numbers of verbal and physical interventions by the participant during the violent argument. The number of physical interventions had a significantly greater mean in the in-group condition compared to the out-group. The more that participants perceived that the Victim was looking to them for help the greater the number of interventions in the in-group but not in the out-group. These results are supported by standard statistical analysis of variance, with more detailed findings obtained by a symbolic regression procedure based on genetic programming. Verbal interventions made during their experience, and analysis of post-experiment interview data suggest that in-group members were more prone to confrontational intervention compared to the out-group who were more prone to make statements to try to diffuse the situation.