Anal Chem. 2026 May 13.
The Golgi apparatus (GA) plays crucial roles in myriad biological processes, and aberrant Golgiphagy has been implicated in a number of pathological settings. Given the lack of suitable imaging tools for Golgiphagy and the propensity of chemical probes to dissipate from stressed organelles, we herein report Golgi-proRed, a pH probe covalently anchored on the GA inner membrane, for imaging of Golgiphagy by red fluorescence triggered upon autophagic delivery of GA into acidic lysosomes. The probe contains a GA-homing entity of blue-to-green fluorescence, a rhodamine-lactam entity that becomes red-emissive in acidic lysosomes, and a moiety of dibenzocyclooctyne (DBCO) for bioorthogonal conjugation with azido-phospholipids metabolically incorporated into the membrane of GA. Upon Golgiphagy, the membrane-anchored probe is codelivered with GA fragments into lysosomes, yielding red fluorescence that serves as the readout of Golgiphagy. The imaging fidelity for Golgiphagy is confirmed by starvation-induced red fluorescence, lysosome-dependent red fluorescence, and lack of such a signal in ATG5-knockout cells deficient in autophagy. With this method, we observed that obvious Golgiphagy occurred in iBMDM cells treated with nigericin and lipopolysaccharides (LPSs) or infected by Listeria monocytogenes. These results show the use of Golgi-proRed for sensitive detection of Golgiphagy in biological events and to screen Golgiphagy-inducing agents.