Fluorogen-activating single-chain antibodies for imaging cell surface proteins

Abstract

Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.

NDPK-D (NM23-H4)-mediated externalization of cardiolipin enables elimination of depolarized mitochondria by mitophagy

“…14 For determination of mitochondrial volume/fragmentation changes, cells were first infected with adenoviral vector expressing mitochondria directed mCerulean/mFAP prior to treatment. Cells were then incubated with 5 nM malachite green to reveal the transgene prior to imaging (thereby defining the mitochondria 58 ) and mounted in a Tokai Hit environmental stage (Tokyo, Japan) in a Nikon TI inverted microscope equipped with a sweptfield confocal head. Three dimensional stacks were collected using a 1.49 NA oil objective with excitation/emission wavelength of 640/700 nm with a Z-space separation of 0.2 micron (20 Z-positions); each stack takes 1 s to collect.…”

A three-state effective Hamiltonian for symmetric cationic diarylmethanes

“…44,60,72 Both these dyes and GFP chromophores display a common binding-dependent fluorescence enhancement associated with the suppression of a common double-bond photoisomerization chemistry in the excited state. [4][5][6][7][73][74][75] Our ongoing work shows that this analogy can also be applied to electronic structure along the photoisomerization coordinates of some of the dyes studied here. It does seem to us now that there is a general physics underlying the excited state behavior in all of these systems, and an opportunity for the development and application of quite general model Hamiltonians for fluorogenic monomethine dyes.…”

“…1,2 Recent interest in these dyes has focused on the environmental sensitivity of their optical response. [3][4][5][6][7][8][9][10][11] In particular, a common binding-dependent fluorescence enhancement makes these dyes useful biological markers. [4][5][6]12 The fluorescence enhancement is attributed to the suppression of a competing double-bond isomerization reaction, the rate of which is influenced by the local viscosity.…”

“…[3][4][5][6][7][8][9][10][11] In particular, a common binding-dependent fluorescence enhancement makes these dyes useful biological markers. [4][5][6]12 The fluorescence enhancement is attributed to the suppression of a competing double-bond isomerization reaction, the rate of which is influenced by the local viscosity. [13][14][15][16] Consistent with their long history of use, the low-energy electronic excitations of dyes such as in Scheme 1 were an early target of quantum mechanical modeling efforts.…”

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

“…The fluorogen is completely nonfluorescent in solution, but on binding to the FAP, its fluorescence activity is increased dramatically (15,000-20,000-fold) (25). The bipartite FAP and cognate fluorogen system enables the selective visualization of proteins at the cell surface through the use of a cellimpermeant fluorogen, MG, fused to an 11-unit polyethylene glycol linker (MG11p).…”