Tethered Fluorogen Assay to Visualize Membrane Apposition in Living Cells

Abstract

We describe proof-of-concept for a novel approach for visualizing regions of close apposition between the surfaces of living cells. A membrane-anchored protein with high affinity for a chemical ligand is expressed on the surface of one set of cells, and the cells are co-cultured with a second set of cells that express a membrane-anchored fluorogen-activating protein (FAP). The co-cultured cells are incubated with a bivalent reagent composed of fluorogen linked to the high-affinity ligand, with the concentration of the bivalent reagent chosen to be less than the binding constant for the FAP-fluorogen pair but greater than the binding constant for the ligand-high-affinity protein pair. In these conditions, strong FAP signal is observed only in regions of close proximity between membranes of the two classes of cell, where high local concentration of fluorogen favors binding to the FAP.

Antibody-Linked Fluorogen-Activating Proteins for Antigen Detection and Cell Ablation

“…Specifically, we used dL5**-cetuximab to label wild-type HaCaTs, which were co-cultured with transgenic scFv1-expressing HEK-293 cells. In these cultures, bright yellow fluorescence was apparent at intercellular contact points (Figure 4B, C), in a manner identical to that previously seen between two transgenic cell lines 28 . When the cells were incubated with separate, unlinked MG and TO1 fluorogens (Figure 4D, E), no fluorescence enhancement at contact sites was observed.…”

“…Another opportunity, which extends results reported here, is to use pairs of antibodyconjugated FAPs to visualize locations where two antigens are in close proximity, as in neural or immunological synapses. FAPs expressed from recombinant genes have already been shown to enable such "trans-TEFLA" experiments in cultured cells 28 ,and, it would be of interest to attempt trans-TEFLA labeling using FAP-tagged antibodies as both partners to investigate intercellular contacts in vitro and in vivo.…”

Illuminating Cellular Biochemistry: Fluorogenic Chemogenetic Biosensors for Biological Imaging

“…A more than 10-fold fluorescence increase from the lower affinity dye MG can be observed at the contact sites between co-cultured cells expressing two different FAPs. [117] FAP-based systems can also act as highly localized photosensitizers with dyes functionalized with heavy atoms, notably by activation of the ROS-generating di-iodinated MG analog. [118] Combined with the different strategies to target such systems to tumor cells [119] , this property shows promising potential for targeted photodamage.…”

Fluorogen-activating proteins: beyond classical fluorescent proteins

“…They later expanded the hybrid system to a new type of biosensor, which provides the opportunity to study multiple trafficking proteins in the same cell 47 . Recently, Jarvik et al 48 developed a novel approach based on FAPs and tethered fluorogen for visualizing regions of close apposition between the surfaces of living cells, which has the potential to provide a real-time readout of the proximity status of the membranes of the two cells. More recently, we and Brönstrup group first applied MG/FAP to study translocation efficiencies of molecular scaffolds designed to transport cargos in bacteria, which provided a general method for investigating the translocation capability of compounds across the membrane of bacterial cells ( Fig.…”