A Quantitative Single-cell Flow Cytometry Assay for Retrograde Membrane Trafficking Using Engineered Cholera Toxin

Abstract

The organization and distribution of proteins, lipids, and nucleic acids in eukaryotic cells is an essential process for cell function. Retrograde trafficking from the plasma membrane to the Golgi and endoplasmic reticulum can greatly modify cell membrane composition and intracellular protein dynamics, and thus typifies a key sorting step. However, methods to efficiently quantify the extent or kinetics of these events are currently limited. Here, we describe a novel quantitative and effectively realtime single-cell flow cytometry assay to directly measure retrograde membrane transport. The assay takes advantage of the well-known retrograde trafficking of cholera toxin engineered with splitfluorescent proteins to generate novel tools for immediate monitoring of intracellular trafficking. This approach will greatly extend the ability to study the underlying biology of intracellular membrane trafficking, and how trafficking systems can adapt to the physiologic needs of different cell types and cell states.

Cholera Toxin as a Probe for Membrane Biology

“…To address these problems, we modified the new split fluorescent protein technologies to link a small fragment of GFP to CTx (via fusion to the A2-chain, termed CTB-mNG2 11 ) [ 10 , 157 ]. The approach led to the development of a novel quantitative and near real-time single-cell flow cytometry assay for retrograde membrane transport driven by CTxB binding to GM1 [ 10 ].…”