β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β₂-microglobulin (β₂m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β₂m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β₂m fibrils did not, however, cause cell death. Instead, fragmented β₂m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.     

The effect of motorboat sound on Australian snapper Pagrus auratus inside and outside a marine reserve

Human‐generated sound affects hearing, movement, and communication in both aquatic and terrestrial animals, but direct natural underwater behavioral observations are lacking. Baited underwater video (BUV) were deployed in near shore waters adjacent to Goat Island in the Cape Rodney–Okakari Point Marine Reserve (protected) or outside the reserve approximately four km south in Mathesons Bay (open), New Zealand to determine the natural behavior of Australian snapper Pagrus auratus exposed to motorboat sound. BUVs worked effectively at bringing fish into video range to assess the effects of sound. The snapper inhabiting the protected area showed no behavioral response to motorboat transits; however, fish in the open zones either scattered from the video frame or decreased feeding activity during boat presence. Our study suggests that motorboat sound, a common source of anthropogenic activity in the marine environment can affect fish behavior differently depending on the status of their habitat (protected versus open).     

Intragenic mapping of the Neurospora pyrimidine-3 locus by functional deletions

Summary On the basis of complementation between polar and non-polar pyrimidine-3 mutants of Neurospora crassa, a physical linear order of the polar mutants within the locus is derived. The basis of this derivation is that a polar mutant is an intragenic functional deletion extending from the site of the mutation to the translationally distal end of the locus.Supported by Public Health Service Research Grant AI-01462.     

Life span and reproductive cost explain interspecific variation in the optimal onset of reproduction

Fitness can be profoundly influenced by the age at first reproduction (AFR), but to date the AFR–fitness relationship only has been investigated intraspecifically. Here, we investigated the relationship between AFR and average lifetime reproductive success (LRS) across 34 bird species. We assessed differences in the deviation of the Optimal AFR (i.e., the species‐specific AFR associated with the highest LRS) from the age at sexual maturity, considering potential effects of life history as well as social and ecological factors. Most individuals adopted the species‐specific Optimal AFR and both the mean and Optimal AFR of species correlated positively with life span. Interspecific deviations of the Optimal AFR were associated with indices reflecting a change in LRS or survival as a function of AFR: a delayed AFR was beneficial in species where early AFR was associated with a decrease in subsequent survival or reproductive output. Overall, our results suggest that a delayed onset of reproduction beyond maturity is an optimal strategy explained by a long life span and costs of early reproduction. By providing the first empirical confirmations of key predictions of life‐history theory across species, this study contributes to a better understanding of life‐history evolution.     

Collapse of an avifauna: climate change appears to exacerbate habitat loss and degradation

Aim  We characterized changes in reporting rates and abundances of bird species over a period of severe rainfall deficiency and increasing average temperatures. We also measured flowering in eucalypts, which support large numbers of nectarivores characteristic of the region.
Location  A 30,000-km² region of northern Victoria, Australia, consisting of limited amounts of remnant native woodlands embedded in largely agricultural landscapes.
Methods  There were three sets of monitoring studies, pitched at regional (survey programmes in 1995–97, 2004–05 and 2006–08), landscape (2002–03 and 2006–07) and site (1997–2008 continuously) scales. Bird survey techniques used a standard 2-ha, 20-min count method. We used Bayesian analyses of reporting rates to document statistically changes in the avifauna through time at each spatial scale.
Results  Bird populations in the largest remnants of native vegetation (up to 40,000 ha), some of which have been declared as national parks in the past decade, experienced similar declines to those in heavily cleared landscapes. All categories of birds (guilds based on foraging substrate, diet, nest site; relative mobility; geographical distributions) were affected similarly. We detected virtually no bird breeding in the latest survey periods. Eucalypt flowering has declined significantly over the past 12 years of drought.
Main conclusions  Declines in the largest woodland remnants commensurate with those in cleared landscapes suggest that reserve systems may not be relied upon to sustain species under climate change. We attribute population declines to low breeding success due to reduced food. Resilience of bird populations in this woodland system might be increased by active management to enhance habitat quality in existing vegetation and restoration of woodland in the more fertile parts of landscapes.     

Recruitment calling: a novel form of extended parental care in an altricial species

In many altricial birds, fledglings disperse when they are no longer fed, and this dispersal marks the end of parental care. In some species, however, young remain in close association with their parents after nutritional independence. Because juveniles are still inferior foragers at this stage, they might benefit from parental assistance in locating good feeding sites, but this possibility remains largely unexplored. Here, we show that parents and helpers in pied babbler (Turdoides bicolor) societies use a recruitment call to direct nutritionally independent, but inexperienced, foragers to particular food patches. Observations and a playback experiment indicated that adult babblers use a "purr" call to recruit group members to a foraging patch. Creation of experimental foraging patches supported observations that individuals tend to give the call when they are foraging on abundant, divisible food sources and when their group contains independent fledglings (youngsters who are no longer fed directly). Fledglings responded to calls more often than adults, who frequently encountered aggression from the caller if they did, and the fledglings gained significant foraging benefits. This is the first study to demonstrate that altricial birds may use recruitment calls to extend parental care past the period of direct provisioning.     

Characterisation of the conformational properties of urea-unfolded Im7: implications for the early stages of protein folding

The colicin immunity protein Im7 folds from its unfolded state in 6 M urea to its native four-helix structure through an on-pathway intermediate that lacks one of the helices of the native structure (helix III). In order to further characterize the folding mechanism of Im7, we have studied the conformational properties of the protein unfolded in 6 M urea in detail using heteronuclear NMR. Triple-resonance experiments with 13C/15N-labelled Im7 in 6 M urea provided almost complete resonance assignments for the backbone nuclei, and measurement of backbone 15N relaxation parameters allowed dynamic ordering of the unfolded polypeptide chain to be investigated. Reduced spectral density mapping and fitting backbone R2 relaxation rates to a polymer dynamics model identified four clusters of interacting residues, each predicted by the average area buried upon folding for each residue. Chemical shift analyses and measurement of NOEs detected with a long mixing-time 1H-1H-15N NOESY-HSQC spectrum confirmed the formation of four clusters. Each cluster of interacting side-chains in urea-unfolded Im7 occurs in a region of the protein that forms a helix in the protein, with the largest clusters being associated with the three long helices that are formed in the on-pathway folding intermediate, whilst the smallest cluster forms a helix only in the native state. NMR studies of a Phe15Ala Im7 variant and a protein in which residues 51-56 are replaced by three glycine residues (H3G3 Im7*), indicated that the clusters do not interact with each other, possibly because they are solvated by urea, as indicated by analysis of NOEs between the protein and the solvent. Based on these data, we suggest that dilution of the chaotrope to initiate refolding will result in collapse of the clusters, leading to the formation of persistent helical structure and the generation of the three-helix folding intermediate.     

The oligomeric state and arrangement of the active bacterial translocon

Protein secretion in bacteria is driven through the ubiquitous SecYEG complex by the ATPase SecA. The structure of SecYEG alone or as a complex with SecA in detergent reveal a monomeric heterotrimer enclosing a central protein channel, yet in membranes it is dimeric. We have addressed the functional significance of the oligomeric status of SecYEG in protein translocation using single molecule and ensemble methods. The results show that while monomers are sufficient for the SecA- and ATP-dependent association of SecYEG with pre-protein, active transport requires SecYEG dimers arranged in the back-to-back conformation. Molecular modeling of this dimeric structure, in conjunction with the new functional data, provides a rationale for the presence of both active and passive copies of SecYEG in the functional translocon.     

Distinguishing Closely Related Amyloid Precursors Using an RNA Aptamer

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human β₂-microglobulin (hβ₂m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hβ₂m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2′F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hβ₂m with an EC₅₀ of ∼200 nm. NMR spectroscopy was used to assign the ¹H-¹⁵N HSQC spectrum of the B6-hβ₂m complex, revealing that the aptamer binds to the face of hβ₂m containing the A, B, E, and D β-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hβ₂m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.