A quantitative single-cell assay for retrograde membrane traffic enables rapid detection of defects in cellular organization.

Retrograde membrane trafficking from plasma membrane to Golgi and endoplasmic reticulum typifies one of the key sorting steps emerging from the early endosome that affects cell surface and intracellular protein dynamics underlying cell function. While some cell surface proteins and lipids are known to sort retrograde, there are few effective methods to quantitatively measure the extent or kinetics of these events. Here we took advantage of the well-known retrograde trafficking of cholera toxin and newly defined split fluorescent protein technology to develop a quantitative, sensitive, and effectively real-time single-cell flow cytometry assay for retrograde membrane transport. The approach can be applied in high throughput to elucidate the underlying biology of membrane traffic and how endosomes adapt to the physiologic needs of different cell types and cell states.