Microenvironment mapping via Dexter energy transfer on immune cells

Abstract

Many disease pathologies can be understood through the elucidation of localized biomolecular networks, or microenvironments. To this end, enzymatic proximity labeling platforms are broadly applied for mapping the wider spatial relationships in subcellular architectures. However, technologies that can map microenvironments with higher precision have long been sought. Here, we describe a microenvironment-mapping platform that exploits photocatalytic carbene generation to selectively identify protein-protein interactions on cell membranes, an approach we term MicroMap (μMap). By using a photocatalyst-antibody conjugate to spatially localize carbene generation, we demonstrate selective labeling of antibody binding targets and their microenvironment protein neighbors. This technique identified the constituent proteins of the programmed-death ligand 1 (PD-L1) microenvironment in live lymphocytes and selectively labeled within an immunosynaptic junction.

Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks

“…iv) Protein labeling via conserved amino acid residues enabling proteome-wide surfaceome interrogations in a broad range of evolutionary distinct organisms. Recently, photocatalysts producing short-lived carbene with nanosecond half-lives were utilized for antibody-guided mapping of protein microdomains that could not be resolved using enzyme-based strategies (intermediates with half-lives of 0.1 ms to 60 s) 27 . Proximity labeling by LUX-MS is mediated by photosensitized singlet oxygen species with a lifetime of 25 -36 μs 43 indicating spatial resolution in between enzyme-and carbene-based approaches.…”

“…Finally, we employed LUX-MS for the spatiotemporal analysis of intercellular surfaceome signaling domains within immunological synapses. Previous attempts were limited to molecular analysis in model systems that use soluble, plate-bound, or membrane-associated factors to mimic immunosynaptic T-cell activation 65 or to fluorescence-based reporting of transient intercellular immune cell interactions 27,79 . We here performed a gp33 immunogen-guided LUX-MS in an isotopically barcoded two-cell system of millions of synchronized and functional immunosynapses and thereby obtained a holistic view on the mature immunosynaptic surfaceome signaling architecture, fostering the formulation of novel biological hypotheses.…”

“…Recently, enzymebased proximity labeling strategies such as APEX 20 , HRP or PUP-iT 21 enabled labeling and MS-based identification of lipid rafts 22 , growth factor signaling domains 23 , B-cell receptor neighborhoods 24 and intact neuronal synapses 25 on living cells and in fixed tissues 26 . A photocatalyst-based proximity labeling strategy thereby showed improved spatial precision 27 . However, in such methods, labeling probes are simultaneously allocated to all surface accessible receptors by antibodies or genetic fusion resulting in a lack of flexibility and specificity to detect ligand-targeted surfaceome signaling domains, leaving the functional hot-spots of the cellular surfaceome landscape unexplored.…”

Boron-Enabled Geometric Isomerization of Small Alkene Fragments via Selective Energy Transfer Catalysis

“…Furthermore, the bio-compatibility of this activation strategy has recently been validated by MacMillan and co-workers (25). Photosensitized alkene isomerization has a venerable history, but efficiency often restricts the scope to styrenyl chromophores (3).…”

Mapping transmembrane binding partners for E-cadherin ectodomains

“…Recently, a micro-mapping platform using a photocatalytic induced carbene was also introduced for proximity labeling of membrane bound proteins [4]. While enormously powerful, these assays merely report on protein proximity; testing direct interactions between the proximate proteins require these techniques to be integrated with a complementary biophysical method.…”