The mechanism of toxicity in HET-S/HET-s prion incompatibility.

The HET-s protein from the filamentous fungus Podospora anserina is a prion involved in a cell death reaction termed heterokaryon incompatibility. This reaction is observed at the point of contact between two genetically distinct strains when one harbors a HET-s prion (in the form of amyloid aggregates) and the other expresses a soluble HET-S protein (96% identical to HET-s). How the HET-s prion interaction with HET-S brings about cell death remains unknown; however, it was recently shown that this interaction leads to a relocalization of HET-S from the cytoplasm to the cell periphery and that this change is associated with cell death. Here, we present detailed insights into this mechanism in which a non-toxic HET-s prion converts a soluble HET-S protein into an integral membrane protein that destabilizes membranes. We observed liposomal membrane defects of approximately 10 up to 60 nm in size in transmission electron microscopy images of freeze-fractured proteoliposomes that were formed in mixtures of HET-S and HET-s amyloids. In liposome leakage assays, HET-S has an innate ability to associate with and disrupt lipid membranes and that this activity is greatly enhanced when HET-S is exposed to HET-s amyloids. Solid-state nuclear magnetic resonance (NMR) analyses revealed that HET-s induces the prion-forming domain of HET-S to adopt the ß-solenoid fold (previously observed in HET-s) and this change disrupts the globular HeLo domain. These data indicate that upon interaction with a HET-s prion, the HET-S HeLo domain partially unfolds, thereby exposing a previously buried ∼34-residue N-terminal transmembrane segment. The liberation of this segment targets HET-S to the membrane where it further oligomerizes, leading to a loss of membrane integrity. HET-S thus appears to display features that are reminiscent of pore-forming toxins.

Freezing continuous-flow self-assembly in a microfluidic device: toward imaging of liposome formation.

A new method is described that combines a microfluidic device for the controlled formation of liposomes with instantaneous immobilization by means of ultrarapid cooling. The microfluidic device is composed of capillaries to hydrodynamically focus a stream of lipids dissolved in 2-propanol by two adjacent aqueous buffer streams before rapidly cooling by propane jet-freezing. The capillary containing the frozen sheath-flow is subsequently separated from the flow-focusing unit and trimmed with cryo-ultramicrotomy for imaging with cryo-scanning electron microscopy (SEM). The emergence of liposomes could be visualized by cryo-SEM without the need for chemical fixation or labeling. We demonstrate that the method is capable of revealing in more detail the formation of nonequilibrium liposomes. Partially and completely formed liposomes were observed at the miscible alcohol-buffer interface. The number density of lipid vesicles varied along the focused interface, and we frequently found clusters of liposomes. Additionally, evidence for the formation of disclike transient intermediates is presented. The method is not limited to studying self-assembly processes only. It can be extended to other biochemical reactions, crystallization processes, and even systematic interfacial mixing studies between different solvents.