TY  - JOUR
TI  - A Quantitative Single-cell Flow Cytometry Assay for Retrograde Membrane Trafficking Using Engineered Cholera Toxin
AU  - Simpson, Mariska S.
AU  - Lencer, Wayne I
AU  - Luong, Phi 
VL  - 10
IS  - 15
PY  - 2020
DA  - 2020/08/05
SP  - e3707
C1  - Bio-protocol 2020;10:e3707
DO  - 10.21769/BioProtoc.3707
UR  - https://doi.org/10.21769/BioProtoc.3707
AB  - 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 real-time 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 split-fluorescent 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.
KW  - Retrograde membrane transport
KW  - Golgi
KW  - Endoplasmic reticulum
KW  - Split fluorescent protein
KW  - Cholera toxin
KW  - Flow cytometry
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False