Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.

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Signaling from the epidermal growth factor receptor (EGFR) via phosphorylation on its C-terminal tyrosine residues requires self-association, which depends on the diffusional properties of the receptor and its density in the plasma membrane. Dimerization is a key event for EGFR activation, but the role of higher order clustering is unknown. We employed single particle tracking to relate the mobility and aggregation of EGFR to its signaling activity. EGFR mobility alternates between short-lived free, confined and immobile states. In the immobile state, EGFR tends to aggregate in clathrin-coated pits, which is further enhanced in a phosphorylation-dependent manner and does not require ligand binding. EGFR phosphorylation is further amplified by cross-phosphorylation in clathrin-coated pits. Because phosphorylated receptors can escape from the pits, local gradients of signaling active EGFR are formed. These results show that amplification of EGFR phosphorylation by receptor clustering in clathrin-coated pits supports signal activation at the plasma membrane.

Supramolecular synthesis represents a flexible approach to the generation of dynamic multicomponent materials with tunable properties. Here, cellular uptake systems based on dynamic supramolecular copolymers have been developed using a combination of differently functionalized discotic molecules. Discotics featuring peripheral amine functionalities that endow the supramolecular polymer with cellular uptake capabilities were readily synthesized. This enabled the uptake of otherwise cell-impermeable discotics via cotransport as a function of supramolecular coassembly. Dynamic multicomponent and multifunctional supramolecular polymers represent a novel and unique platform for modular cellular uptake systems.

Self-assembly of discotic molecules into supramolecular polymers offers a flexible approach for the generation of multicomponent one-dimensional columnar architectures with tuneable biomedical properties. Decoration with ligands induces specific binding of the self-assembled scaffold to biological targets. The modular design allows the easy co-assembly of different discotics for the generation of probes for targeted imaging and cellular targeting with adjustable ligand density and composition.