The accumulation of amyloid ␤ peptide(1-42) (A␤(1-42)) in extracellular plaques is one of the pathological hallmarks of Alzheimer disease (AD). Several studies have suggested that cellular reuptake of A␤(1-42) may be a crucial step in its cytotoxicity, but the uptake mechanism is not yet understood. A␤ may be present in an aggregated form prior to cellular uptake. Alternatively, monomeric peptide may enter the endocytic pathway and conditions in the endocytic compartments may induce the aggregation process. Our study aims to answer the question whether aggregate formation is a prerequisite or a consequence of A␤ endocytosis. We visualized aggregate formation of fluorescently labeled A␤(1-42) and tracked its internalization by human neuroblastoma cells and neurons. ␤-Sheet-rich A␤(1-42) aggregates entered the cells at low nanomolar concentration of A␤(1-42). In contrast, monomer uptake faced a concentration threshold and occurred only at concentrations and time scales that allowed A␤(1-42) aggregates to form. By uncoupling membrane binding from internalization, we found that A␤(1-42) monomers bound rapidly to the plasma membrane and formed aggregates there. These structures were subsequently taken up and accumulated in endocytic vesicles. This process correlated with metabolic inhibition. Our data therefore imply that the formation of ␤-sheet-rich aggregates is a prerequisite for A␤(1-42) uptake and cytotoxicity.One of the pathological hallmarks of Alzheimer disease (AD) 2 is the presence of extracellular plaques composed mainly of 42-amino acid amyloid ␤ peptide (A␤(1-42)) (1). The small hydrophobic A␤(1-42) peptide, which is generated by proteolytic cleavage of the amyloid precursor protein, is released as a monomer from the plasma membrane into extracellular space, and tends to aggregate spontaneously into oligomeric, protofibrillar, and fibrillar assemblies (2-4). Oligomeric species of A␤(1-42) are tightly linked to AD pathogenesis and are presumed to be the cause of neuronal damage (5). Several studies have suggested that the reuptake of extracellular A␤(1-42) into neurons may lead to the formation of intracellular aggregates, resulting in neuronal damage and neurotoxicity (6 -8). Endocytosis of misfolded proteins has also been observed in cell models of the tau protein, ␣-synuclein and huntingtin (9, 10), and recent evidence suggests that it may be the initial step in the replication of the misfolded protein structures by prion mechanisms (10 -14). Several possible endocytic pathways, such as macropinocytosis and receptor-mediated endocytosis, have been discussed for A␤ and other misfolded protein aggregates (15-19). However, our understanding of the connection between aggregation and cytotoxicity is still limited. It has not been conclusively determined how and when the A␤(1-42) peptide becomes toxic, whether A␤ aggregates prior to internalization or during the internalization process and, if so, in which intracellular compartments the aggregates form. Elucidating the connection between aggregation and i...

The influence of cholesterol's alkyl side chain on membrane properties was studied using a series of synthetic cholesterol derivatives without a side chain or with a branched side chain consisting of 5 to 14 carbon atoms. Cholesterol's side chain is crucial for all membrane properties investigated and therefore essential for the membrane properties of eukaryotic cells.

a b s t r a c tCellular pathways involving a-synuclein (aS) seem to be causative for development of Parkinson's disease. Interactions between aS and lipid membranes appear to be important for the physiological function of the protein and influence the pathological aggregation of aS leading to the formation of amyloid plaques. Upon membrane binding the unstructured aS folds into amphipathic helices. In our work we characterized the penetration depth and probed the local environment of Trp-residues introduced along the aS sequence. We could show that while the entire helix is well embedded in the lipid bilayer, segments with a shallower penetration and supposable higher flexibility exist.

The genome of influenza A viruses (IAV) is encoded in eight distinct viral ribonucleoproteins (vRNPs) that consist of negative sense viral RNA (vRNA) covered by the IAV nucleoprotein. Previous studies strongly support a selective packaging model by which vRNP segments are bundling to an octameric complex, which is integrated into budding virions. However, the pathway(s) generating a complete genome bundle is not known. We here use a multiplexed FISH assay to monitor all eight vRNAs in parallel in human lung epithelial cells. Analysis of 3.9 × 10 5 spots of colocalizing vRNAs provides quantitative insights into segment composition of vRNP complexes and, thus, implications for bundling routes. The complexes rarely contain multiple copies of a specific segment. The data suggest a selective packaging mechanism with limited flexibility by which vRNPs assemble into a complete IAV genome. We surmise that this flexibility forms an essential basis for the development of reassortant viruses with pandemic potential.