Fig 1.
Live-cell labeling strategies described in this work.
(A) Structure of a CoA-reporter conjugate [7], and examples of possible reporter dyes, easily attachable through maleimide chemistry to the thiol group of CoA. (B) Labeling of small peptide tags, genetically fused to the protein of interest (POI), mediated through 4′-phosphopantetheinyl transferase (PPTase) enzymes. (C) A HaloTag ligand (HTL), comprising a chloroalkane, polyethylene glycol (PEG) linker units for better access to the catalytic center of the enzyme as well as for improved solubility [8], and a reporter, in this example again coupled via thiol chemistry as in (A).The HaloTag, which can be genetically fused to the protein of interest, is a haloalkane dehalogenase engineered to stably form a reaction intermediate, allowing covalent incorporation of labeled substrates.
Fig 2.
Non-specific staining of cells due to coupling of CoA derivatives to cytoplasmic molecules.
(A, B) Schemes illustrating the labeling protocols with wash steps performed either on cells in suspension (A) or on adherent cells (B), respectively. (C) Flow cytometry reveals that non-specific coupling of CoA-Atto488 is, although still present, massively reduced by washing HEK 293 cells while still adherent. (D) Confocal microscopy illustrates homogenous cytosolic stain after washing HEK 293 cells in suspension, indicating cytoplasmic uptake and strong retention of label. Predominantly membranous labeling is observed if CoA-488 is removed while tagged HER2-expressing cells are still adherent. (E) Two-step labeling, in which CoA-biotin on the surface of HEK 293 cells is detected in flow cytometry by streptavidin-Alexa Fluor 647, results only in very limited non-specific staining, in addition to some generic streptavidin binding to the cell surface. (F) CoA-DY 647, a far-red dye, results in significant background binding, irrespective of the optimized wash protocol in flow cytometry.
Fig 3.
Comparison of PPTase and HaloTag (HT) labeling.
(A) Using cell-permeable HTL-TMR, saturation is reached by labeling for 15 min with nanomolar concentrations with high specificity (vermillion curve), as very low signal is found in the absence of HT fusion (bluish green curve). (B) In contrast, saturation is not reached by 5 μM of cell-permeable CoA-SiR, even after 30 min incubation with 1 μM Sfp enzyme (vermillion), with about 30% of the signal being non-specific (in the absence of enzyme, bluish green). (C) A HaloTag ligand (HTL) based on Alexa Fluor 660 results in extremely low non-specific signal (absence of HaloTag, inset, bluish green), while the kinetics are only slightly less favorable than those for HTL-TMR (vermillion). (D) Flow cytometry of HEK 293 cells (no HT, no Sfp enzyme) (black), compared with the same cells after non-specific labeling after 15 min incubation with 15 μM of the respective HTL (blue) or CoA derivative (vermillion).