The endoplasmic reticulum (ER) has undeniable fundamental importance in protein post-translational modifications in cells. Proteins gain their final three-dimensional folding in the ER lumen. Therefore it is imperative to check protein aggregation in ER since in numerous pathological conditions including neurodegenerative disease, diabetes, cancer, stroke, preeclampsia, heart disease and viral infections, ER homeostasis can be destroyed easily.
Recently, fluorophores with aggregation-induced emission features (AIEgens) have been considerably developed. AIEgens seem attractive to have a clear cellular image due to their capability to light up remarkably in aggregation state in opposition to conventional fluorophores. This characteristic could be beneficial to study cellular response or behavior in both live and fixed cells in different conditions.
In this study, we used our designed AIEgen dyes to track protein aggregation in ER lumen especially in stress conditions in human cells. Firstly non-cell toxicity of dyes has been proved for HeLa cell line by Alamar Blue test. More than 90 percent of stained cells with dyes were alive in comparison with unstained cells population. Also, the concentration and incubation time have been optimized through using flow cytometry. The designed dye has three parts, a fluorescent part with the ability to recognize cysteine in unfolded proteins in live cells that conjugated to a peptide. The peptide sequence is excellent for ER targeting. So, the synthesized dye has a considerable ability to trace unfolded or aggregated proteins in ER lumen which could be beneficial to early detection or monitoring drug effects on the disease process. The function of ER targeting peptide has been checked by comparing this fluorescent sensor with fluorescent dyes in similar structure but without the peptide. Confocal microscopy and flow cytometry data show that this peptide is not only live cell permeable but it also can sense unfolded protein in ER successfully.