FORTIS: a live-cell assay to monitor AMPA receptors using pH-sensitive fluorescence tags

The real-time live fluorescent monitoring of surface AMPA receptors (AMPARs) could open new opportunities for drug discovery and phenotypic screening concerning neuropsychiatric disorders. We have developed FORTIS, a tool based on pH sensitivity capable of detecting subtle changes in surface AMPARs at a neuronal population level. The expression of SEP-GluA1 or pHuji-GluA1 recombinant AMPAR subunits in mammalian neurons cultured in 96-well plates enables surface AMPARs to be monitored with a microplate reader. Thus, FORTIS can register rapid changes in surface AMPARs induced by drugs or genetic modifications without having to rely on conventional electrophysiology or imaging. By combining FORTIS with pharmacological manipulations, basal surface AMPARs, and plasticity-like changes can be monitored. We expect that employing FORTIS to screen for changes in surface AMPARs will accelerate both neuroscience research and drug discovery.

Non-invasive and label-free 3D-visualization shows in vivo oligomerization of the staphylococcal alkaline shock protein 23 (Asp23)

Fluorescence-tags, commonly used to visualize the spatial distribution of proteins within cells, can influence the localization of the tagged proteins by affecting their stability, interaction with other proteins or the induction of oligomerization artifacts. To circumvent these obstacles, a protocol was developed to generate 50 nm thick serial sections suitable for immunogold labeling and subsequent reconstruction of the spatial distribution of immuno-labeled native proteins within individual bacterial cells. Applying this method, we show a cellular distribution of the staphylococcal alkaline shock protein 23 (Asp23), which is compatible with filament formation, a property of Asp23 that we also demonstrate in vitro.

Microhomology-based CRISPR tagging tools for protein tracking, purification, and depletion

Work in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relies on overexpression of tagged proteins because high-quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals. Here we describe a versatile toolset for rapid tagging of endogenous proteins. The strategy utilizes CRISPR/Cas9 and microhomology-mediated end joining repair for efficient tagging. We provide tools to insert 3×HA, His6FLAG, His6-Biotin-TEV-RGSHis6, mCherry, GFP, and the auxin-inducible degron tag for compound-induced protein depletion. This approach and the developed tools should greatly facilitate functional analysis of proteins in their native environment.

Smartly designed AIE triazoliums as unique targeting fluorescence tags for sulfonic biomacromolecule recognition via ‘electrostatic locking’

Smartly designed AIE triazoliums as unique targeting fluorescence tags for specific sulfonic biomacromolecule recognition via ‘electrostatic locking’.

Cell uptake, intracellular distribution, fate and reactive oxygen species generation of polymer brush engineered CeO2−xNPs

Cerium Oxide nanoparticles are modified with spherical brushes of charged polyelectrolytes to study the effect of the surface charge on their biological fate and to shield fluorescence tags.

Engineered fluorescence tags for in vivo protein labelling

In vivoprotein labelling with a peptide tag–fluorescent probe system is an important chemical biology strategy for studying protein distribution, interaction and function.