Abstract
The Golgi apparatus is central to intracellular trafficking, yet its dynamic visualization remains constrained by probes that require high concentrations and long incubations. Here we present cycling molecular assemblies (CyMA), a design concept that harnesses endogenous dynamic enzymatic futile cycle to drive ultrasensitive imaging. The BODIPY-CyMA probe operates through reversible palmitoylation-depalmitoylation mediated by palmitoyl acyltransferases and thioesterases, establishing a nonequilibrium steady-state that actively concentrates the probe at the Golgi. This enzymatic cycling converts diffusion-limited localization into self-amplifying signal generation, enabling morphology imaging at concentrations as low as 100 pM and real-time tracking of Golgi dynamics within minutes at nanomolar levels. This probe requires minimal incubation time, exhibits negligible cytotoxicity, faithfully reports pharmacological Golgi disassembly, and functions in vivo in Drosophila larvae. BODIPY-CyMA exemplifies how coupling molecular self-assembly to endogenous enzymatic cycles affords a general strategy for constructing dynamic, non-perturbative probes for live cell and in vivo imaging.