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 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.
The influenza A virus (IAV) genome is segmented into eight viral ribonucleoproteins, each expressing a negatively oriented viral RNA (vRNA). Along the infection cycle, highly abundant single-stranded small viral RNAs (svRNA) are transcribed in a segment-specific manner. The sequences of svRNAs and of the vRNA 5'-ends are identical and highly conserved among all IAV strains. Here, we demonstrate that these sequences can be used as a target for a pan-selective sensor of IAV infection. To this end, we used a complementary fluorescent forced-intercalation RNA (IAV QB-FIT) probe with a single locked nucleic acid substitution to increase brightness. We demonstrated by fluorescence in situ hybridization (FISH) that this probe is suitable and easy to use to detect infection of different cell types by a broad variety of avian, porcine, and human IAV strains, but not by other influenza virus types. IAV QB-FIT also provides a useful tool to characterize different infection states of the host cell.
and found that the recovery times of dCas9:sgRNA complexes on the target were about 6, 12 and 60 min. for a 7mer, 8mer and 11mer, respectively. This suggests that the complexes having shorter sgRNA seeds exchange with the target at a higher rate, implying a weaker affinity. We also compared the PAM sequences AG vs. GG, with the same length of the sgRNA seed sequence. The fluorescence recovery times were the same (> 60 min.), indicating similar affinities to the target and that the reported lower cleavage efficiency of AG PAM is possibly due to its inefficient targeting specificity. Our use of dCas9 in these in vivo studies has allowed us to deconvolve the overall CRISPR cycle to extract the DNA binding step and further to facilitate the optimization of Cas9-based gene editing.
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