Optical Trapping with High Forces Reveals Unexpected Behaviors of Prion Fibrils

Amyloid fibrils are important in diverse cellular functions, feature in many human diseases and have potential applications in nanotechnology. Here we describe methods that combine optical trapping and fluorescent imaging to characterize the forces that govern the integrity of amyloid fibrils formed by a yeast prion protein. A crucial advance was to use the self-templating properties of amyloidogenic proteins to tether prion fibrils, enabling their manipulation in the optical trap. At normal pulling forces the fibrils were impervious to disruption. At much higher forces (up to 250 pN), discontinuities occurred in force-extension traces before fibril rupture. Experiments with selective amyloid-disrupting agents and mutations demonstrated that such discontinuities were caused by the unfolding of individual subdomains. Thus, our results reveal unusually strong noncovalent intermolecular contacts that maintain fibril integrity even when individual monomers partially unfold and extend fibril length.National Institutes of Health (U.S.) (Grant GM025874)National Science Foundation (U.S.). CAREER (Award 0643745

Neural dynamics of shooting decisions and the switch from freeze to fight

Real-life shooting decisions typically occur under acute threat and require fast switching between vigilant situational assessment and immediate fight-or-flight actions. Recent studies suggested that freezing facilitates action preparation and decision-making but the neurocognitive mechanisms remain unclear. We applied functional magnetic resonance imaging, posturographic and autonomic measurements while participants performed a shooting task under threat of shock. two independent studies, in unselected civilians (N = 22) and police recruits (N = 54), revealed that preparation for shooting decisions under threat is associated with postural freezing, bradycardia, midbrain activity (including the periaqueductal gray-PAG) and PAG-amygdala connectivity. Crucially, stronger activity in the midbrain/pAG during this preparatory stage of freezing predicted faster subsequent accurate shooting. Finally, the switch from preparation to active shooting was associated with tachycardia, perigenual anterior cingulate cortex (pgACC) activity and pgACC-amygdala connectivity. These findings suggest that threat-anticipatory midbrain activity centred around the PAG supports decision-making by facilitating action preparation and highlight the role of the pgACC when switching from preparation to action. These results translate animal models of the neural switch from freeze-to-action. In addition, they reveal a core neural circuit for shooting performance under threat and provide empirical evidence for the role of defensive reactions such as freezing in subsequent action decision-making

[SWI+], the Prion Formed by the Chromatin Remodeling Factor Swi1, Is Highly Sensitive to Alterations in Hsp70 Chaperone System Activity

The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments

Modulation of Aβ(42 )low-n oligomerization using a novel yeast reporter system

BACKGROUND: While traditional models of Alzheimer's disease focused on large fibrillar deposits of the Aβ(42 )amyloid peptide in the brain, recent work suggests that the major pathogenic effects may be attributed to SDS-stable oligomers of Aβ(42). These Aβ(42 )oligomers represent a rational target for therapeutic intervention, yet factors governing their assembly are poorly understood. RESULTS: We describe a new yeast model system focused on the initial stages of Aβ(42 )oligomerization. We show that the activity of a fusion of Aβ(42 )to a reporter protein is compromised in yeast by the formation of SDS-stable low-n oligomers. These oligomers are reminiscent of the low-n oligomers formed by the Aβ(42 )peptide in vitro, in mammalian cell culture, and in the human brain. Point mutations previously shown to inhibit Aβ(42 )aggregation in vitro, were made in the Aβ(42 )portion of the fusion protein. These mutations both inhibited oligomerization and restored activity to the fusion protein. Using this model system, we found that oligomerization of the fusion protein is stimulated by millimolar concentrations of the yeast prion curing agent guanidine. Surprisingly, deletion of the chaperone Hsp104 (a known target for guanidine) inhibited oligomerization of the fusion protein. Furthermore, we demonstrate that Hsp104 interacts with the Aβ(42)-fusion protein and appears to protect it from disaggregation and degradation. CONCLUSION: Previous models of Alzheimer's disease focused on unravelling compounds that inhibit fibrillization of Aβ(42), i.e. the last step of Aβ(42 )assembly. However, inhibition of fibrillization may lead to the accumulation of toxic oligomers of Aβ(42). The model described here can be used to search for and test proteinacious or chemical compounds for their ability to interfere with the initial steps of Aβ(42 )oligomerization. Our findings suggest that yeast contain guanidine-sensitive factor(s) that reduce the amount of low-n oligomers of Aβ(42). As many yeast proteins have human homologs, identification of these factors may help to uncover homologous proteins that affect Aβ(42 )oligomerization in mammals

Biosurfactant production by Arctic and Antarctic bacteria growing on hydrocarbons

The risk of hydrocarbon contamination in marine polar areas is constantly increasing. Autochthonous bacteria, due to their ability to cope and survive under extreme environmental conditions, can play a fundamental role in the hydrocarbon degradation. The degradation process is often enhanced by the production of biosurfactant molecules. The present study reports for the first time on the isolation of biosurfactant-producing bacteria from Arctic and Antarctic shoreline sediments. A total of 199 psychrotolerant bacterial isolates were obtained from hydrocarbon-amended (with crude or diesel oil) microcosms. A total of 18 isolates were selected for their ability to grow in the presence of crude oil and produce biosurfactants, as it was revealed by the production of good E24 values (≥50 %) and/or reduction in the surface tension (under 30 mN/m). The positive response of the isolates to both tests suggests a possible production of biosurfactants with emulsifying and interfacial activities. Biosurfactant-producing isolates were mainly affiliated to the genera Rhodococcus (14 isolates), followed by Pseudomonas (two isolates), Pseudoalteromonas (one isolate) and Idiomarina (one isolate). Thin-layer chromatography of biosurfactant crude extracts revealed that the majority of the selected isolates were able to produce glycolipidic surfactants. Our results enlarge the knowledge, which is still poor and fragmentary, on biosurfactant producers from polar areas and indicate marine polar sediments as a source of bacteria with potential applications in the remediation of hydrocarbon-contaminated cold environments