AbstractBackgroundAmyloidâbeta (AÎČ), is generated by sequential cleavage of the amyloid precursor protein (APP) by betaâ and gammaâsecretase. The intracellular sites of cleavage are unknown due to the lack of tools that can detect de novo AÎČ production.MethodUsing unnatural amino acid (UAA) mutagenesis and click chemistry, a single fluorophore (magenta) siteâspecifically binds to AÎČ within the APP construct. The Câterminus of APP is fused with the green fluorescent protein mClover, allowing for a distinction between fullâlength APP and AÎČ, as the uncleaved APP will appear white (magenta+green) from the bifluorescence, contrasting with the magenta of AÎČ alone when separated from the green mClover Câterminus. Live cells expressing the APPâmClover construct (green) were incubated with cellâimpermeable tetrazineâAlexaFluor 594 (magenta) at 4°C, which clickâlabeled the UAA on AÎČ on cell surface APP. The cells were then incubated at 37°C, resuming endocytosis of bifluorescent APP.ResultAfter clickâlabeling, the APP construct resides exclusively at the cell surface. Once warmed to 37°C, the intracellular bifluorescence increases with time, suggesting APP endocytosis (Fig 1). Using objectâbased analyses, the magenta clickâlabel:green mClover ratio was determined from superâresolution zâstack images (Fig 2). The magenta:green ratio was lower in cells treated with gammaâsecretase inhibitor (GSI).ConclusionA qualitative and semiâquantitative picture can be created of AÎČâs localization by following the separation of the bifluorescent signal into intraluminal magenta clickâlabel and membraneâbound green mClover. This technology will be coupled with immunocytochemistry of subcellular markers, which will allow for further specificity on the location of AÎČ travel within a cell, with regards to the endolysosomal pathway. Furthermore, this technology can be applied to examine AÎČ within the unique cytoarchitecture of primary neurons which may help inform the design of new therapeutics in this cell type central to AD pathogenesis.