Carbon Dot Blinking Fingerprint Uncovers Native Membrane Receptor Organizations via Deep Learning

Oligomeric
organization of G protein-coupled receptors is proposed
to regulate receptor signaling and function, yet rapid and precise
identification of the oligomeric status especially for native receptors
on a cell membrane remains an outstanding challenge. By using blinking
carbon dots (CDs), we now develop a deep learning (DL)-based blinking
fingerprint recognition method, named deep-blinking fingerprint recognition
(BFR), which allows automatic classification of CD-labeled receptor
organizations on a cell membrane. This DL model integrates convolutional
layers, long–short-term memory, and fully connected layers
to extract time-dependent blinking features of CDs and is trained
to a high accuracy (∼95%) for identifying receptor organizations.
Using deep blinking fingerprint recognition, we found that CXCR4 mainly
exists as 87.3% monomers, 12.4% dimers, and <1% higher-order oligomers
on a HeLa cell membrane. We further demonstrate that the heterogeneous
organizations can be regulated by various stimuli at different degrees.
The receptor-binding ligands, agonist SDF-1α and antagonist
AMD3100, can induce the dimerization of CXCR4 to 33.1 and 20.3%, respectively.
In addition, cytochalasin D, which inhibits actin polymerization,
similarly prompts significant dimerization of CXCR4 to 30.9%. The
multi-pathway organization regulation will provide an insight for
understanding the oligomerization mechanism of CXCR4 as well as for
elucidating their physiological functions.