Epidermal growth factor receptor (EGFR) is a type I receptor tyrosine kinase, the deregulation of which has been implicated in a variety of human carcinomas. EGFR signalling is preceded by receptor dimerization, typically thought to result from a ligand-induced conformational change in the ectodomain that exposes a loop (dimerization arm) required for receptor association. Ligand binding may also trigger allosteric changes in the cytoplasmic domain of the receptor that is crucial for signalling. Despite these insights, ensemble-averaging approaches have not determined the precise mechanism of receptor activation in situ. Using quantum-dot-based optical tracking of single molecules combined with a novel time-dependent diffusivity analysis, here we present the dimerization dynamics of individual EGFRs on living cells. Before ligand addition, EGFRs spontaneously formed finite-lifetime dimers kinetically stabilized by their dimerization arms. The dimers were primed both for ligand binding and for signalling, such that after EGF addition they rapidly showed a very slow diffusivity state that correlated with activation. Although the kinetic stability of unliganded dimers was in principle sufficient for EGF-independent activation, ligand binding was still required for signalling. Interestingly, dimers were enriched in the cell periphery in an actin- and receptor-expression-dependent fashion, resulting in a peripheral enhancement of EGF-induced signalling that may enable polarized responses to growth factors.
High-affinity transferrin receptor (TfR) bispecific antibodies facilitate trafficking of TfR to lysosomes and induce TfR degradation to decrease the ability of TfR to mediate BBB transcytosis.
Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a
nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form
can improve cell-permeability and allow creation of “fluorogenic”
compounds—ligands that shift to the fluorescent zwitterion upon binding a
biomolecular target. An archetype fluorogenic dye is the far-red
tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful
labels for advanced microscopy. Here, we develop a quantitative framework for the
development of new fluorogenic dyes, determining that the lactone–zwitterion
equilibrium constant (KL–Z) is sufficient to predict
fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded
new fluorescent and fluorogenic labels with improved performance in cellular imaging
experiments. We then designed a novel fluorophore—Janelia Fluor 526
(JF526)—with SiR-like properties but shorter fluorescence excitation
and emission wavelengths. JF526 is a versatile scaffold for fluorogenic
probes including ligands for self-labeling tags, stains for endogenous structures, and
spontaneously blinking labels for super-resolution immunofluorescence. JF526
constitutes a new label for advanced microscopy experiments, and our quantitative
framework will enable the rational design of other fluorogenic probes for
bioimaging.
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