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

Abstract

Retrograde membrane trafficking from plasma membrane to the Golgi and endoplasmic reticulum affects intracellular protein dynamics underlying cell function. Here, we developed split-fluorescent toxin reporters that enable a quantitative, sensitive, and real-time single-cell flow cytometry assay for retrograde membrane transport.

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

“…Kinetic time course experiments on retrograde trafficking across diverse conditions and cell lines can be reliably quantified by flow cytometry. Notably, we obtained high levels of reproducibility in K562 cells with calculated Z-factors of 0.92 and 0.96 for TGN and ER transport, respectively (Luong et al, 2020). A Z-factor > 0.5 is considered ideal for high-throughput screening (Zhang et al, 1999).…”

“…The assay depends on CTB's ability to bind GM1 at the cell membrane and the C-terminal KDEL-motif of the CTA2-chain's ability to bind the ER-retention KDEL-receptor (Lencer and Tsai, 2003). Additionally, we applied the split fluorescent protein (spilt-FP) technologies to CTx for single-cell flow cytometry (Feng et al, 2017;Luong et al, 2020). The CTA2 chain was engineered to include the split monomeric neon green2 (mNG211) peptide and was coexpressed in E. coli with the homopentameric CTB.…”

“…The assay also requires cell-lines of interest to stably express the complementary portion of the split neon green2 fragment (mNG21-10) into organelle compartments of the retrograde pathway. We recently engineered reporter systems to monitor retrograde trafficking from the plasma membrane into the Trans-Golgi Network (TGN) and also the Endoplasmic Reticulum (ER) (Luong et al, 2020). Various cell lines including adherent epithelial cells (e.g., human embryonic kidney (HEK 293T) and African Green Monkey kidney COS7) and cells grown in suspension (e.g., human lymphocyte K562 cells) have been validated for retrograde TGN and ER retrograde trafficking (Luong et al, 2020).…”

“…We recently engineered reporter systems to monitor retrograde trafficking from the plasma membrane into the Trans-Golgi Network (TGN) and also the Endoplasmic Reticulum (ER) (Luong et al, 2020). Various cell lines including adherent epithelial cells (e.g., human embryonic kidney (HEK 293T) and African Green Monkey kidney COS7) and cells grown in suspension (e.g., human lymphocyte K562 cells) have been validated for retrograde TGN and ER retrograde trafficking (Luong et al, 2020). Some cell lines may not have GM1 lipid and thus will normally not be suitable for the toxin reporter assay.…”

Structural basis for acyl chain control over glycosphingolipid sorting and vesicular trafficking

“…For these studies, the different GM1 species were applied to a HeLa cell line that endogenously lacks GM1. CTxB trafficking into the ER was quantitatively measured using a FACS-based split-GFP assay recently developed by our group (60). We found that only the short chain (≤ C14:0) or unsaturated GM1 species (≤ C22:1 Δ13 ), both lacking the C14*-motif, efficiently trafficked CTxB into the ER (Fig.…”