Live Cell Imaging: Tips and Tools

Melanie M. Frigault, Judith Lacoste, Jody L. Swift, Claire M. Brown
2009 Biophysical Journal  
Alzheimer's disease (AD) is linked to the self-association of amyloid-b peptide (Ab), a protein of 39-43 amino acids that is normally soluble in the plasma and cerebrospinal fluid. Although large, fibrillar aggregates were long thought to be the pathogenic agents, recent evidence indicates that soluble Ab oligomers are more closely linked to disease progression. In fact, negative effects have been observed from oligomers as small as dimers and trimers. A number of compounds have been found to
more » ... hibit the large-scale aggregation of Ab in bulk solution, typically by manipulating the b-sheet structure characteristic of these assemblies, but little is known regarding inhibition of the earliest association steps. We have used single-molecule fluorescence spectroscopy to characterize the efficacy of four known peptide-based inhibitors toward preventing or reversing association in the earliest Ab oligomers (n ¼ 2-5). Fluoresceinand biotin-labeled Ab(1-40) is tethered to functionalized cover slips (pM concentrations) through biotin-streptavidin binding. Spatially resolved monomers and oligomers are examined, one at a time; the number of associated peptides in each species is determined based on quantized photobleaching of the individual dye molecules. Distributions of Ab monomers and oligomers are determined through examination of dozens of individual peptide species, and permit comparison of the different inhibitor compounds. Results will be presented for inhibition under neutral versus acidic conditions (pH 7.4 versus 5.8). Collectively, these studies will provide new insight into the potential for reversing or preventing Ab association in its earliest stages.
doi:10.1016/j.bpj.2008.12.047 fatcat:xqfbsxaazzgwricfr5jsgmqvf4